Novel card proteins involved in cell death regulation

ABSTRACT

The present invention provides NB-ARC and CARD-containing proteins (NACs), nucleic acid molecules encoding NACs and antibodies specific for at least one NAC. The invention further provides chimeric NAC proteins. The invention also provides screening assays for identifying an agent that can effectively alter the association of a NAC with a NAC-associated protein. The invention further provides methods of modulating apoptosis in a cell by introducing into the cell a nucleic acid molecule encoding a NAC or an antisense nucleotide sequence. The invention also provides a method of using a reagent that can specifically bind to a NAC to diagnose a pathology that is characterized by an increased or decreased level of apoptosis in a cell.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to the fields of molecularbiology and molecular medicine and more specifically to theidentification of proteins involved in programmed cell death andassociations of these proteins.

[0003] 2. Background Information

[0004] Programmed cell death is a physiologic process that ensureshomeostasis is maintained between cell production and cell turnover inessentially all self-renewing tissues. In many cases, characteristicmorphological changes, termed “apoptosis,” occur in a dying cell. Sincesimilar changes occur in different types of dying cells, cell deathappears to proceed through a common pathway in different cell types.

[0005] In addition to maintaining tissue homeostasis, apoptosis alsooccurs in response to a variety of external stimuli, including growthfactor deprivation, alterations in calcium levels, free-radicals,cytotoxic lymphokines, infection by some viruses, radiation and mostchemotherapeutic agents. Thus, apoptosis is an inducible event thatlikely is subject to similar mechanisms of regulation as occur, forexample, in a metabolic pathway. In this regard, dysregulation ofapoptosis also can occur and is observed, for example, in some types ofcancer cells, which survive for a longer time than corresponding normalcells, and in neurodegenerative diseases where neurons die prematurely.In viral infections, induction of apoptosis can figure prominently inthe pathophysiology of the disease process, because immune-basederadication of viral infections depends on elimination ofvirus-producing host cells by immune cell attack resulting in apoptosis.

[0006] Some of the proteins involved in programmed cell death have beenidentified and associations among some of these proteins have beendescribed. However, additional apoptosis regulating proteins remain tobe found and the mechanisms by which these proteins mediate theiractivity remains to be elucidated. The identification of the proteinsinvolved in cell death and an understanding of the associations betweenthese proteins can provide a means for manipulating the process ofapoptosis in a cell and, therefore, selectively regulating the relativelifespan of a cell or its relative resistance to cell death stimuli.

[0007] The principal effectors of apoptosis are a family ofintracellular proteases known as Caspases, representing an abbreviationfor Cysteine Aspartyl Proteases. Caspases are found as inactive zymogensin essentially all animal cells. During apoptosis, the caspases areactivated by proteolytic processing at specific aspartic acid residues,resulting in the production of subunits that assemble into an activeprotease typically consisting of a heterotetramer containing two largeand two small subunits (Thornberry and Lazebnik, Science 281:1312-1316(1998)). The phenomenon of apoptosis is produced directly or indirectlyby the activation of caspases in cells, resulting in the proteolyticcleavage of specific substrate proteins. Moreover, in many cases,caspases can cleave and activate themselves and each other, creatingcascades of protease activation and mechanisms for “auto”-activation.

[0008] Among the substrates of caspases are the intracellular proformsof cytokines such as pro-Interleukin-1β (pro-IL-1β) and pro-IL-18. Whencleaved by caspases, these pro-proteins are converted to thebiologically active cytokines which are then liberated from cells,circulating in the body and eliciting inflammatory immune reactions.Thus, caspases can be involved, in some instances, in cytokineactivation and responses to infectious agents, as well as inflammatoryand autoimmune diseases. Caspases also participate in signaltransduction pathways activated by some cytokine receptors, particularlymembers of the Tumor Necrosis Factor (TNF) family of cytokine receptorswhich are capable of activating certain caspase zymogens.

[0009] Thus, knowledge about the proteins having domains that interactwith and regulate caspases is important for devising strategies formanipulating cell life and death in therapeutically useful ways. Theidentification of such proteins that contain caspase-interacting domainsand the elucidation of the proteins with which they interact, therefore,can form the basis for strategies designed to modulate apoptosis,cytokine production, cytokine receptor signaling, and other cellularprocesses. Thus a need exists to identify proteins that interact withcaspases and other apoptosis related proteins. The present inventionsatisfies this need and provides additional advantages as well.

SUMMARY OF THE INVENTION

[0010] In accordance with the present invention, there are providednovel “NB-ARC and CARD”-containing proteins, designated NAC, as well asseveral isoforms of NAC produced by alternative mRNA splicing. Theinvention also provides nucleic acid molecules encoding NAC and itsisoforms, vectors containing these nucleic acid molecules and host cellscontaining the vectors. The invention also provides antibodies that canspecifically bind to NAC proteins, including alternative isoformsthereof.

[0011] The present invention also provides a screening assay useful foridentifying agents that can effectively alter the association of NACwith itself or with other proteins. By altering the self-association ofNAC or by altering their interactions with other proteins, an effectiveagent may increase or decrease the level of caspase proteolytic activityor apoptosis in a cell, or it may increase or decrease the levels ofNF-κB, cytokine production, or other events.

[0012] The invention also provides methods of altering the activity ofNAC in a cell, wherein such increased or decreased activity of NAC canmodulate the level of apoptosis or other cellular responses. Forexample, the activity of NAC in a cell can be increased by introducinginto the cell and expressing a nucleic acid sequence encoding theseproteins. In addition, the activity of NAC in a cell can be decreased byintroducing into the cell and expressing a fragment of NAC, or anantisense nucleotide sequence that is complementary to a portion of anucleic acid molecule encoding the NAC proteins.

[0013] The invention also provides methods for using an agent that canspecifically bind NAC or a nucleotide sequence that can bind to anucleic acid molecule encoding NAC to diagnose a pathology that ischaracterized by an altered level of apoptosis due to an increased ordecreased level of NAC in a cell.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1A shows the cloning strategy for NAC and Isoforms of NAC.The NB-ARC domain (filled box), leucine-rich repeats (LRR, filled bars),and the CARD domain (labeled box) are depicted. Relevant restrictionsites (RI for EcoRI, X for Xho I) are indicated. Positions for forwardPCR primers (1F, 2F, and 3F) and reverse primers (1R, 2R, and 3R) whichwere used for Reverse Transcriptase-Polymerase Chain Reaction cloning ofNAC and NAC-isoforms are shown.

[0015]FIG. 1B shows multiple isoforms of NAC. Isoforms of NAC aregenerated by alternative mRNA splicing, based on cDNA cloning results.The same symbols as in FIG. 1A are used. Two alternatively spliced exonsare shown as dotted boxes and hatched boxes, respectively. Note thatlonger and shorter versions of the CARD domain are produced (CARD_(L)and CARD_(S)). The four resultant isoforms are described as NACα, NACβ,NACγ and NACδ.

[0016]FIG. 1C shows the cDNA and amino acid sequence of the longest NACisoform (also set for in SEQ ID NOs:1 and 2). The nucleotide sequencesof the two alternatively spliced exons (nucleotides 2870-2959, and3784-3915, respectively, and amino acids 918-947 and 1262-1305) areunderlined. The positions for the P-loop (Walker A) and Walker B ofNB-ARC domain are indicated. The LRR repeats are in bold letters (aminoacids 808-948), and the CARD domain is in bold underlined letters (aminoacids 1373-1473).

[0017]FIG. 1D shows a sequence analysis of NAC:NB-ARC homology.Alignment of the NB-ARC domains of human NAC (amino acids 329-547),CARD4 (amino acids 197-408), and Apaf-1 (amino acids 138-352), andCaenorhabditis elegans CED4 (amino acids 154-374). Alignment wasconducted using Clustal method (Thompson et al., Nuc. Acids Res.22:4673-4680 (1994)). Identical and similar residues are shown in blackand gray, respectively.

[0018]FIG. 1E shows alignment of CARD domain of NAC and otherCARD-containing proteins. Alignment was conducted using Clustal method.Identical and similar residues are shown in black and gray,respectively.

[0019]FIG. 2 shows self-association of Long and Short CARD domains ofNAC. (A) For in vitro binding assays, purified GST fusion proteinsimmobilized on GSH-sepharose containing CARD_(L) (lane 3), CARD_(S)(lane 4), or GST alone (lane 2) were incubated with ³⁵S-labeled, invitro translated CARD_(L) (top panel), CARD_(S) (middle panel), orcontrol protein Skp-1 (bottom panel). In vitro translation mix (onetenth of input, lane 1) was directly loaded as control. (B) Homophilicinteractions of CARD. In vitro translated Apaf-1 (-WD) (top panel), CED4(middle panel), or control Skp-1 (bottom panel) proteins were incubatedwith GST (lane 2), GST-CARD_(L) (lane 3), and GST-CARD_(S) (lane 4)immobilized on GSH-sepharose beads. In lane 1, one tenth of input ³⁵Sproteins are shown.

[0020]FIG. 3 shows homophilic interactions of CARD domains detected byyeast two-hybrid method. Yeast cells were co-transformed with plasmidsencoding the indicated proteins fused to LexA DNA binding domain (LexA)and proteins fused to B42 transactivation domain (B42). Transformantswere replica-plated on leucine-supplemented plates (Leu+) andleucine-deficient plates (Leu−) to assess protein interactions.β-galactosidase activity (LacZ) was measured for each transformant, andwere scaled as: absent (−), weak (+/−), detectable (+), strong (++),very strong (+++), and strongest (++++)

[0021]FIG. 4 shows self-association of NB-ARC domain of NAC. In vitrotranslated, ³⁵S-labeled rat reticulocyte lysates (1 μl) containingNB-ARC (lanes 2 and 3) or Skp-1 (as a control; lanes 5 and 6) wereincubated with purified GST-NB-ARC (lanes 3 and 6) or GST alone (lanes 2and 5) immobilized on GSH-sepharose beads for in vitro binding assays.In lanes 1 and 4, one tenth of input ³⁵S proteins are shown.

[0022]FIG. 5 shows that NAC forms complexes with Apaf-1 and CED4. (A)Complex formation with human Apaf-1. 293T cells were transientlytransfected with an expression plasmid encoding HA-tagged human Apaf-1lacking the C-terminal WD repeats [HA-Apaf-1 (ΔWD)] in the presence(lanes 2 and 3) or absence (lane 1) of a plasmid encoding myc-taggedfull-length NAC (myc-NAC). Transfected cells were lysed and subjected toimmunoprecipitation (IP) with either a mouse monoclonal antibody to myc(lanes 1 and 3) or a control mouse IgG (lane 2). Proteins from theimmune complexes were resolved by SDS-PAGE, transferred tonitrocellulose, and subjected to immunoblot analysis (WB) using anti-HAantibodies (bottom panel) followed by anti-myc antibodies (top panel).One tenth of the total cell lysates derived from each transfection wereloaded directly in the gel as a control (Lysate). (B) Complex formationwith C. elegans CED4 protein. Identical procedures and conditionsdescribed for Apaf-1 in (A) were employed for CED4 interaction studieswith NAC.

[0023]FIG. 6 shows that NAC interacts with pro-Casp8, but not pro-Casp9.(A) Interaction with pro-Casp8. 293T cells were transfected with anexpression plasmid encoding HA-tagged human pro-Casp8 [HA-Casp8 (C/A)],which harbors an alanine replacement of the catalytic cysteine residue,in the presence (lanes 2 and 3) or absence (lane 1) of myc-NACexpression plasmid. Transfected cells were lysed and subjected toimmunoprecipitation (IP) with either anti-myc antibodies (lanes 1 and 3)or a control antibody (lane 2). The immunoprecipitated proteins wereresolved by SDS-PAGE, transferred to nitrocellulose, and analyzed byimmunoblotting (WB) for pro-Casp8 (bottom panel) using anti-HAantibodies or for NAC (top panel) using anti-myc antibodies. One tenthof the total cell lysates of each transfection was loaded directly ingels as a control (Lysate). (B) Interaction with pro-Casp9. Identicalprocedures and conditions described for Casp8 were used for Casp9interaction studies with NAC. The Casp9 expression plasmid [Flag-Casp9(C/A)] contains a C-terminal Flag-tagged form of pro-Casp9 harboring analanine replacement of the catalytic cysteine residue. The immunoblotswere probed for Casp9 (bottom panel) using a rabbit anti-Casp9polyclonal antibody derived against GST-Casp9 fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In accordance with the present invention, there are provided“substantially pure” mammalian CARD-containing proteins, designated NACand CARD-X. As used herein, the term “NAC” refers to a protein thatcontains both an NB-ARC domain and a CARD domain (NAC). The inventionNAC proteins represent novel members of the “CARD domain” family ofproteins, which family includes CED-4 and Apaf-1. An invention NACcomprises a NB-ARC domain and a CARD domain, and optionally furthercomprises a leucine-rich repeat domain and/or a TIM-Barrel-like domain.

[0025] As used herein, the term “CARD domain” refers to a CaspaseRecruitment Domain (Hofmann et al., Trends Biochem. Sci. 22:155-156(1997)). CARD domains have been found in some members of the Caspasefamily of cell death proteases. Caspases-1, 2, 4, 5, 9, and 11 containCARD domains near their NH₂-termini. These CARD domains mediateinteractions of the zymogen inactive forms of caspases with otherproteins which can either activate or inhibit the activation of theseenzymes. For example, the CARD domain of pro-caspase-9 binds to the CARDdomain of a caspase-activating protein called Apaf-1 (Apoptosis ProteaseActivating Factor-1). Similarly, the CARD domain of pro-caspase-1permits interactions with another CARD protein known as Cardiac (alsoreferred to as RIP2 and RICK), which results in activation of thecaspase-1 protease (Thome et al., Curr. Biol. 16:885-888 (1998)). And,pro-caspase-2 binds to the CARD protein Raidd (also know as Cradd),which permits recruitment of pro-caspase-2 to Tumor Necrosis Factor(TNF) Receptor complexes and which results in activation of thecaspase-2 protease (Ahmad et al., Cancer Res. 57:615-619 (1997)). CARDdomains can also participate in homotypic interactions with themselves,resulting in self-association of proteins that contain theseprotein-interaction domains and producing dimeric or possibly evenoligomeric complexes.

[0026] CARD domains can be found in association with other types offunctional domains within a single polypeptide, thus providing amechanism for bringing a functional domain into close proximity orcontact with a target protein via CARD:CARD associations involving twoCARD-containing proteins. For example, the Caenorhabiditis elegans celldeath gene ced-4 encodes a protein that contains a CARD domain and aATP-binding oligomerization domain called an NB-ARC domain (van derBiezen and Jones Curr Biol 8:R226-R227). The CARD domain of the CED-4protein interacts with the CARD domain of a pro-caspase called CED-3.The NB-ARC domain allows CED-4 to self-associate, thereby forming anoligomeric complex which brings associated pro-CED-3 molecules intoclose proximity to each other. Because most pro-caspases possess atleast a small amount of protease activity even in their unprocessedform, the assembly of a complex that brings the proforms of caspase intojuxtaposition can result in trans-processing of zymogens, producing theproteolytically processed and active caspase. Thus, CED-4 employs a CARDdomain for binding a pro-caspase and an NB-ARC domain forself-oligomerization, resulting in caspase clustering, proteolyticprocessing and activation.

[0027] Numerous CED-4-related proteins have recently been identified.These proteins belong to the CED-4 family of proteins, and include CED-4(Yuan and Horvitz, Development 116:309-320 (1992)), Apaf-1, (Zou et al.,Cell 90:405-413 (1997)), Dark (Rodriguez et al., Nature Cell Biol.1:272-279 (1999)), and CARD4/Nod1 (Bertin et al., J. Biol. Chem.274:12955-12958 (1999) and Inohara et al., J. Biol. Chem.274:14560-14567 (1999)). As used herein, a CED-4 family member is aprotein that comprises a NB-ARC domain and a CARD domain.

[0028] The CED-4 homolog in humans and rodents, referred to as Apaf-1,has been found to function similarly. The Apaf-1 protein contains a (i)CARD domain, (ii) NB-ARC domain, and (iii) multiple copies of aWD-repeat domain. In contrast to CED-4 which can spontaneouslyoligomerize, the mammalian Apaf-1 protein is an inactive monomer untilinduced to oligomerize by binding of a co-factor protein, cytochrome c(Li et al., Cell 91:479-489 (1997)). In Apaf-1, the WD repeat domainsprevent oligomerization of the Apaf-1 protein, until coming into contactwith cytochrome c. Thus, the WD-repeats function as anegative-regulatory domain that maintains Apaf-1 in a latent state untilcytochrome c release from damaged mitochondria triggers the assembly ofan oligomeric Apaf-1 complex (Saleh, J. Biol. Chem. 274:17941-17945(1999)). By binding pro-caspase-9 through its CARD domain, Apaf-1oligomeric complexes are thought to bring the zymogen forms of caspase-9into close proximity, permitting them to cleave each other and producethe proteolytic processed and active caspase-9 protease (Zou et al., J.Biol. Chem. 274:11549-11556 (1999)).

[0029] In addition to their role in caspase-activation, CARD domainshave been implicated in other cellular processes. Some CARD-containingproteins, for example, induce activation of the transcription factorNF-κB. NF-κB activation is induced by many cytokines and plays animportant role in cytokine receptor signal transduction mechanisms(DiDonato et al., Nature 388:548-554 (1997)). Moreover, CARD domains arefound in some proteins that inhibit rather than activate caspases, suchas the IAP (Inhibitor of Apoptosis Protein) family members, cIAP1 andcIAP2 (Rothe et al., Cell 83:1243-1252 (1995)) and oncogenic mutants ofthe Bcl-10 protein (Willis et al., Cell 96:35-45 (1999)). Also, thoughcaspase activation resulting from CARD domain interactions is ofteninvolved in inducing apoptosis, other caspases are primarily involved inproteolytic processing and activation of inflammatory cytokines (such aspro-IL-1β and pro-IL-18). Thus, CARD-containing proteins can also beinvolved in cytokine production, thus regulating immune and inflammatoryresponses.

[0030] In view of the function of the CARD domain within invention NACproteins, invention NAC proteins or CARD-domain containing fragmentsthereof, are contemplated herein for use in methods to modulateapoptosis, cytokine production, cytokine receptor signaling, and othercellular processes. Invention NAC proteins or CARD-domain containingfragments thereof are also contemplated in methods to identifyCARD-binding agents that modulate apoptosis, cytokine production,cytokine receptor signaling, and other cellular processes.

[0031] In one embodiment, a CARD domain of an invention NAC comprises asequence with at least 50% identity to the CARD domain of NAC (see,e.g., residues 1373-1473 of SEQ ID NO:2). More preferably, a CARD domainof the invention comprises a sequence with at least 60% identity to theCARD domain of NAC. Most preferably, a CARD domain of the inventioncomprises a sequence with at least 75% identity to the CARD domain ofNAC. Typically, a CARD domain of the invention comprises a sequence withat least 95% identity to the CARD domain of NAC.

[0032] As described herein, invention NAC or CARD-X proteins canassociate with other CARD-containing proteins. In particular, theassociation of the CARD domain of invention NAC proteins with anotherCARD-containing protein, such as Apaf-1, CED-4, caspases-1, 2, 9, 11,cIAPs-1 and 2, CARDIAK, Raidd, Dark, CARD4, and other NAC or CARD-X, andthe like, is sufficiently specific such that the bound complex can formin vivo in a cell or in vitro under suitable conditions. Similarlytherefore, an invention NAC protein can associate with another NACprotein by CARD:CARD association.

[0033] A NAC protein of the invention further can associate withpro-caspases, caspases or with caspase-associated proteins, therebymodulating caspase proteolytic activity. Caspase proteolytic activity isassociated with apoptosis of cells, and additionally with cytokineproduction. Therefore, an invention NAC can modulate apoptosis orcytokine production by modulating caspase proteolytic activity. As usedherein a “caspase” is any member of the cysteine aspartyl proteases thatassociates with a NAC protein of the invention or with a NAC associatedprotein. Similarly, a “pro-caspase” is an inactive or less-activeprecursor form of a caspase, which is typically converted to the moreactive caspase form by a proteolytic event.

[0034] CARD-containing proteins are also known to induce activation ofthe transcription factor NF-κB. Thus, an invention NAC can also modulatetranscription by modulation of NF-κB activity.

[0035] A NAC protein of the invention also comprises a NB-ARC domain. Asdescribed herein, a NB-ARC domain of the invention NAC protein comprisesa sequence wherein the identity of residues in either the P-Loop (WalkerA) or Walker B regions is at least 60% relative to the residues of NAC(see, e.g., residues 329-343 and 407-412 of SEQ ID NO:2; see FIG. 1C).Preferably, an NB-ARC domain of the invention NAC comprises a sequencewherein the overall identity of residues in the P-Loop (Walker A) andWalker B regions is at least 60% relative to the residues of NAC. Morepreferably, an NB-ARC domain of the invention comprises a sequence withat least 60% identity to the entire NB-ARC domain of NAC (see, e.g.,residues 329-547 of SEQ ID NO:2). Most preferably, an NB-ARC domain ofthe invention comprises a sequence with at least 80% identity to theentire NB-ARC domain of NAC.

[0036] The NB-ARC domain of the invention NAC proteins associates withother proteins, particularly with proteins comprising NB-ARC domains.Thus, a functional NB-ARC domain associates with NB-ARCdomain-containing proteins by way of NB-ARC:NB-ARC association. As usedherein, the term “associate” or “association” means that NAC can bind toa protein relatively specifically and, therefore, can form a boundcomplex. In particular, the association of the NB-ARC domain of NAC withanother NB-ARC domain-containing proteins is sufficiently specific suchthat the bound complex can form in vivo in a cell or in vitro undersuitable condition. Further, a NB-ARC domain demonstrates bothnucleotide-binding (e.g., ATP-binding) and hydrolysis activities, whichis typically required for its ability to associate with NB-ARCdomain-containing proteins. Thus, an NB-ARC domain of the invention NACcomprises one or more nucleotide binding sites. As used herein, anucleotide binding site is a portion of a protein that specificallybinds a nucleotide such as, e.g., ATP, and the like. Typically, thenucleotide binding site of NB-ARC will comprise a P-loop, a kinase 2motif, or a kinase 3a motif of the invention NAC (these motifs aredefined, for example, in van der Biezen and Jones, supra). Preferably,the nucleotide binding site of NB-ARC comprises a P-loop of theinvention NAC.

[0037] An invention NAC, therefore, is capable of CARD:CARD associationand/or NB-ARC:NB-ARC association, resulting in a multifunctional proteincapable of one or more specific associations with other proteins. Aninvention NAC can modulate cell processes such as apoptosis, cytokineproduction, and the like. For example, it is contemplated herein that aninvention NAC protein can increase the level of apoptosis in a cell. Itis also contemplated herein that an invention NAC can decrease the levelof apoptosis in a cell. For example, a NAC which does not induceapoptosis may form hetero-oligomers with a NAC which is apoptotic, thusinterfering with the apoptosis-inducing activity of NAC.

[0038] In another embodiment of the invention the NAC protein of theinvention also contains Leucine-Rich Repeats (LRR) domain, similar to aLRR described in another CARD protein known as CARD4 (also known asNod1) (Inohara et al., J. Biol. Chem. 274:14560-14567 (1999)). UnlikeCARD-4 (Nod1), however, the CARD domain of NAC is located at theCarboxyl end of the protein whereas the CARD domain of CARD-4 (Nod1) isfound at the NH₂-end of the protein. The function of the LRR domain isto mediate specific interactions with other proteins.

[0039] As used herein, leucine-rich repeat (LRR) domain of the inventionNAC comprises a sequence with at least 50% identity to the LRR domain ofNAC (see, e.g., residues 808-948 of SEQ ID NO:2). Preferably, a LRRdomain of the invention NAC comprises a sequence with at least 60%identity to the LRR domain of NAC. More preferably, a LRR region of theinvention NAC comprises a sequence with at least 75% identity to the LRRdomain of NAC. Most preferably, a LRR region of the invention NACcomprises a sequence with at least 95% identity to the LRR domain ofNAC.

[0040] It is further contemplated herein that a shortened LRR of theinvention NAC may be used. A shortened LRR of the invention comprises asequence with at least 90% identity to the splice variant form of theLRR (see, e.g., residues 808-917 of SEQ ID NO:2), and does not containmore than 90% of the residues in the splice region (see, e.g., residues918-947 of SEQ ID NO:2). Preferably, the shortened LRR does not containmore than 70% of the residues in the splice region. More preferably, theshortened LRR does not contain more than 50% of the residues in thesplice region. The shortened LRR will be of particular utility when theprotein:protein interaction activity of a NAC comprising a shortened LRRdiffers from that observed for a NAC comprising the full-length LRR.Activity of a NAC with a shortened LRR will be determined by one or moreof the assays disclosed herein, and shall be considered to differ fromthat of a NAC comprising the full-length LRR if any protein:proteininteractions are altered by 10% or more, or if caspase activity orapoptotic activity is altered by 10% or more.

[0041] In a further embodiment of the invention, invention NAC proteinscontain a TIM-Barrel-like domain with similarity to TIM-barrel proteins.TIM-Barrel domains are well known in the art and typically consist ofeight alternating α-helices and β-strands forming a barrel-likestructure, but may contain 7 α-helices and/or β-strands in someinstances. TIM-barrels have been found in some enzymes, such asaldolase, but also mediate protein interactions in some instances.

[0042] As used herein, a TIM-Barrel-like domain of an invention NACcomprises a sequence with at least 50% identity to the TIM-Barrel-likedomain of NAC (residues 1079-1320 of SEQ ID NO:2). Preferably, aTIM-barrel-like domain of the invention NAC comprises a sequence with atleast 60% identity to the TIM-Barrel-like domain of NAC. Morepreferably, a TIM-barrel domain of the invention NAC comprises asequence with at least 70% identity to the TIM-barrel-like domain ofNAC. Most preferably, a TIM-barrel-like domain of the invention NACcomprises a sequence with at least 80% identity to the TIM-barrel-likedomain of NAC.

[0043] Presently preferred NAC proteins of the invention includeproteins that comprise substantially the same amino acid sequences asthe protein sequence set forth in SEQ ID NOs:2, 4, and 6, as well asbiologically active, functional fragments thereof.

[0044] Those of skill in the art will recognize that numerous residuesof the above-described sequences can be substituted with other,chemically, sterically and/or electronically similar residues withoutsubstantially altering the biological activity of the resulting NACprotein species. In addition, larger polypeptide sequences containingsubstantially the same sequence as amino acids set forth in SEQ IDNOs:2, 4, and 6, therein are contemplated.

[0045] As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptides (or nucleic acids referred to hereinbefore)containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions, or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

[0046] The term “biologically active” or “functional”, when used hereinas a modifier of invention NACs, or polypeptide fragments thereof,refers to a polypeptide that exhibits functional characteristics similarto a NAC. Biological activities of NAC are, for example, the ability tobind, preferably in vivo, to a CARD-containing protein or aNB-ARC-containing protein, or to homo-oligomerize, or to modulateprotease activation, particularly caspase activation, or to modulateNF-κB activity, or to modulate apoptosis, as described herein. Such NACbinding activity can be assayed, for example, using the methodsdescribed herein. Another biological activity of NAC is the ability toact as an immunogen for the production of polyclonal and monoclonalantibodies that bind specifically to an invention NAC. Thus, aninvention nucleic acid encoding NAC will encode a polypeptidespecifically recognized by an antibody that also specifically recognizesa NAC protein (preferably human) including the amino acid set forth inSEQ ID NOs:2, 4, 6, 10 or 12. Such immunologic activity may be assayedby any method known to those of skill in the art. For example, atest-polypeptide encoded by a NAC cDNA can be used to produceantibodies, which are then assayed for their ability to bind to aninvention NAC protein including the sequence set forth in SEQ ID NOs:2,4, 6, 10 or 12. If the antibody binds to the test-polypeptide and theprotein including the sequence encoded by SEQ ID NOs:2, 4, 6, 10 or 12with substantially the same affinity, then the polypeptide possesses therequisite immunologic biological activity.

[0047] As used herein, the term “substantially purified” means a proteinthat is in a form that is relatively free from contaminating lipids,proteins, nucleic acids or other cellular material normally associatedwith a protein in a cell. A substantially purified NAC can be obtainedby a variety of methods well-known in the art, e.g., recombinantexpression systems described herein, precipitation, gel filtration,ion-exchange, reverse-phase and affinity chromatography, and the like.Other well-known methods are described in Deutscher et al., Guide toProtein Purification: Methods in Enzymology Vol. 182, (Academic Press,(1990)), which is incorporated herein by reference. Alternatively, theisolated polypeptides of the present invention can be obtained usingwell-known recombinant methods as described, for example, in Sambrook etal., supra., (1989).

[0048] In addition to the ability of invention NAC proteins, orfragements thereof, to interact with other, heterologous proteins (i.e.,NB-ARC and CARD-containing proteins), invention NAC and CARD-X proteinshave the ability to self-associate. This self-association is possiblethrough interactions between CARD domains, and also through interactionsbetween NB-ARC domains. Further, self-association can take place as aresult of interactions between LRR and TIM-Barrel-like domains.

[0049] In accordance with the invention, there are also providedmutations and fragments of NAC which have activity different than a wildtype NAC activity. As used herein, a “mutation” can be any deletion,insertion, or change of one or more amino acids in the wild type proteinsequence, and a “fragment” is any truncated form, eithercarboxy-terminal, amino-terminal, or both, of the wild type protein.Preferably, the different activity of the mutation or fragment is aresult of the mutant protein or fragment maintaining some but not all ofthe activities of wild type NAC. For example, a fragment of NAC cancontain a CARD domain and LRR and TIM-Barrel-like domains, but lack afunctional NB-ARC domain. Such a fragment will maintain a portion of thewild type NAC activity (e.g., CARD domain functionality), but not allwild type activities (e.g., lacking an active NB-ARC domain). Theresultant fragment will therefore have activity different than wild typeNAC activity. In one embodiment, the activity of the fragment will be“dominant negative.” A dominant negative activity will allow thefragment to reduce or inactivate the activity of one or more isoforms ofwild type NAC.

[0050] Isoforms of the NAC proteins are also provided which arise fromalternative mRNA splicing and may alter or modify the interactions ofthe NAC protein with other proteins. For example, three novel isoformsof NAC are provided herein and designated: NACβ, NACγ and NACδ (setforth as SEQ ID Nos:1, 3 and 5, respectively). The amino acid sequenceand the portion of cDNA encoding the amino acid sequence of NACβ isshown in FIG. 1C, and the NACβ cDNA and amino acid sequences are listedas SEQ ID NOs: 1 and 2, respectively. NACβ represents the NAC splicevariant in which both splice regions are present in the translatedpolypeptide, thereby including the nucleic acids 1-4422 of the NAC cDNAsequence and amino acids 1-1473 of the NAC protein sequence of FIG. 1C.NACγ represents the NAC splice variant in which neither splice region ispresent in the translated polypeptide, thereby including the nucleicacids 1-2869, 2960-3783, and 3916-4422 of the NAC cDNA sequence andamino acids 1-917, 948-1261, and 1306-1473 of the NAC protein sequenceof FIG. 1C. The NACγ cDNA and amino acid sequences are listed as SEQ IDNOs:3 and 4, respectively. NACδ represents the NAC splice variant inwhich only the more carboxy-terminal splice region is present in thetranslated polypeptide, thereby including the nucleic acids 1-2869, and2960-4422 of the NAC cDNA sequence and amino acids 1-917, and 948-1473of the NAC protein sequence of FIG. 1C. The NACδ cDNA and amino acidsequences are listed as SEQ ID NOs:5 and 6, respectively.

[0051] In another embodiment of the invention, chimeric proteins areprovided comprising NAC, or a functional fragment thereof, fused withanother protein or functional fragment thereof. Functional fragments ofNAC include, for example, NB-ARC, CARD, LRR and TIM-Barrel-like domains,as defined herein. Proteins with which the NAC or functional fragmentthereof are fused will include, for example, glutathione-S-transferase,an antibody, or other proteins or functional fragments thereof whichfacilitate recovery of the chimera. Further proteins with which the NACor functional fragment thereof are fused will include, for example,luciferase, green fluorescent protein, an antibody, or other proteins orfunctional fragments thereof which facilitate identification of thechimera. Still further proteins with which the NAC or functionalfragment thereof are fused will include, for example, the LexA DNAbinding domain, ricin, α-sarcin, an antibody, or other proteins whichhave therapeutic properties or other biological activity.

[0052] Further invention chimeric proteins contemplated herein arechimeric proteins wherein a domain of the NAC is replaced by a similarsuch domain from a heterologous protein. For example, the NB-ARC domainof NAC, as described above, can be replaced by the NB-ARC domain ofApaf-1, and the like. Another example of such a chimera is a proteinwherein the CARD domain of NAC is replaced by the CARD domain fromCED-4, and the like.

[0053] The CARD-X protein contains a CARD domain and a region withsimilarity to TIM-Barrel-like domains, but otherwise is distinct fromNAC. The cDNA sequence encoding CARD-X (SEQ ID NO:7) reveals that itarises from a separate gene from NAC. The predicted CARD-X amino acidsequence (SEQ ID NO:8), in particular, does not contain an NB-ARCdomain.

[0054] A CARD domain of the CARD-X protein comprises a sequence with atleast 50% identity to the CARD domain of CARD-X (residues 343-431 of SEQID NO:8). More preferably, a CARD domain of the invention comprises asequence with at least 60% identity to the CARD domain of CARD-X. Mostpreferably, a CARD domain of the invention comprises a sequence with atleast 75% identity to the CARD domain of CARD-X. Typically, a CARDdomain of the invention comprises a sequence with at least 95% identityto the CARD domain of CARD-X.

[0055] A TIM-Barrel-like domain of CARD-X comprises a sequence with atleast 50% identity to the TIM-Barrel domain of CARD-X (residues 56-331of SEQ ID NO:8). Preferably, a TIM-barrel domain of the invention NACcomprises a sequence with at least 60% identity to the TIM-Barrel domainof CARD-X. More preferably, a TIM-barrel domain of the invention CARD-Xcomprises a sequence with at least 70% identity to the TIM-barrel domainof CARD-X. Most preferably, a TIM-barrel domain of the CARD-X comprisesa sequence with at least 80% identity to the TIM-barrel domain ofCARD-X.

[0056] In one embodiment, invention chimeric CARD-containing proteinsprovided herein are designated NAC-X. Nucleic acids that encode NAC-Xare also provided herein. Alternative isoforms of the NAC-X proteins andthe corresponding nucleic acids that encode the alternative isoforms arealso provided. As used herein, the term “NAC-X” refers to chimericproteins comprising portions of a NAC and portions of CARD-X. Forexample, one type of NAC-X protein is a NACδ-X, wherein a portion ofNACδ, for example, the TIM-Barrel-like domain of NACδ, is replaced by aportion of CARD-X, for example, the TIM-Barrel-like domain of CARD-X. Itis within the scope of this invention that a protein comprising portionsof a domain common to both NAC and CARD-X, particularly the CARD andTIM-Barrel-like domains, can comprise a chimera of NAC and CARD-X. Forexample, a NACβ-X protein can have residues 1-1397 from SEQ ID NO:2immediately followed by residues 364-402 from SEQ ID NO:8, which are inturn immediately followed by residues 1436-1473 from SEQ ID NO:2, thusforming a chimeric CARD domain.

[0057] In one embodiment, a NAC-X protein will comprise an NB-ARC domainof NAC, as previously described, and the CARD domain of CARD-X. Inanother embodiment, a NAC-X protein will comprise the NB-ARC domain andLRR domain of NAC, the CARD domain of CARD-X, and the TIM-Barrel-likedomain from either NAC or CARD-X or a chimera from both. In yet anotherembodiment, NAC-X will comprise the NB-ARC and LRR domains of NAC andthe CARD and TIM-Barrel-like domains of CARD-X. For example, inventionchimeric proteins can include residues between 1-947 and 1-1078 of NACβ(SEQ ID NO:2) or between 1-918 and 1-1048 of NACγ or NACδ (SEQ ID NOs:4and 6, respectively) in chimera with residues between 1-431 and 56-431of CARD-X (SEQ ID NO:8). A particular invention chimera is termed NAC-X1a protein, and comprises the following sequences: NACβ-X1, residues1-1078 of NACβ and residues 56-431 of CARD-X, having the resultant aminoacid sequence listed in SEQ ID NO:10; NACγ/δ-X1 residues 1-1048 of NACγor NACδ and residues 56-431 of CARD-X, having the resultant amino acidsequence listed in SEQ ID NO:12. The cDNA encoding NACβ-X1 comprisescDNA residues 1-3234 of NACβ and 166-1293 of CARD-X, having theresultant sequence listed in SEQ ID NO:9; and the cDNA encodingNACγ/δ-X1 proteins comprise cDNA residues 1-3144 of NACγ or NACδ and166-1293 of CARD-X, having the resultant sequence listed in SEQ IDNO:11.

[0058] Another embodiment of the invention provides NAC, or a functionalfragment thereof, fused with a moiety to form a conjugate. As usedherein, a “moiety” can be a physical, chemical or biological entitywhich contributes functionality to NAC or a functional fragment thereof.Functionalities contributed by a moiety include therapeutic or otherbiological activity, or the ability to facilitate identification orrecovery of NAC. Therefore, a moiety will include molecules known in theart to be useful for detection of the conjugate by, for example, byfluorescence, magnetic imaging, detection of radioactive emission. Amoiety may also be useful for recovery of the conjugate, for example aHis tag or other known tags used for protein isolation/purification, ora physical substance such as a bead. A moiety can be a therapeuticcompound, for example, a cytotoxic drug which can be useful to effect abiological change in cells to which the conjugate localizes.

[0059] An example of the means for preparing the inventionpolypeptide(s) is to express nucleic acids encoding the NAC in asuitable host cell, such as a bacterial cell, a yeast cell, an amphibiancell (i.e., oocyte), or a mammalian cell, using methods well known inthe art, and recovering the expressed polypeptide, again usingwell-known methods. Invention polypeptides can be isolated directly fromcells that have been transformed with expression vectors as describedbelow herein. The invention polypeptide, biologically functionalfragments, and functional equivalents thereof can also be produced bychemical synthesis. For example, synthetic polypeptides can be producedusing Applied Biosystems, Inc. Model 430A or 431A automatic peptidesynthesizer (Foster City, Calif.) employing the chemistry provided bythe manufacturer.

[0060] Also encompassed by the term NAC are functional fragments orpolypeptide analogs thereof. The term “functional fragment” refers to apeptide fragment that is a portion of a full length NAC protein,provided that the portion has one or more biological activities, asdefined above, that is characteristic of the corresponding full lengthNAC. For example, a functional fragment of an invention NAC protein canhave one or more of the protein:protein binding activities prevalent inNAC. In addition, the characteristic of a functional fragment ofinvention NAC proteins to elicit an immune response is useful forobtaining an anti-NAC antibodies. Thus, the invention also providesfunctional fragments of invention NAC proteins, which can be identifiedusing the binding and routine methods, such as bioassays describedherein.

[0061] The term “polypeptide analog” includes any polypeptide having anamino acid residue sequence substantially the same as a sequencespecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the ability to functionally mimic an NAC as described herein.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another.

[0062] The amino acid length of functional fragments or polypeptideanalogs of the present invention can range from about 5 amino acids upto the full-length protein sequence of an invention NAC. In certainembodiments, the amino acid lengths include, for example, at least about10 amino acids, at least about 20, at least about 30, at least about 40,at least about 50, at least about 75, at least about 100, at least about150, at least about 200, at least about 250 or more amino acids inlength up to the full-length NAC protein sequence.

[0063] As used herein the phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue, provided that such polypeptide displays therequired binding activity. The phrase “chemical derivative” refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Such derivatized moleculesinclude, for example, those molecules in which free amino groups havebeen derivatized to form amine hydrochlorides, p-toluene sulfonylgroups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatized to formsalts, methyl and ethyl esters or other types of esters or hydrazides.Free hydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-im-benzylhistidine. Also included as chemical derivatives arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For examples:4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine. Polypeptides of the present invention alsoinclude any polypeptide having one or more additions and/or deletions ofresidues, relative to the sequence of a polypeptide whose sequence isshown herein, so long as the required activity is maintained.

[0064] The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified NAC mature proteinor functional polypeptide fragments thereof, alone or in combinationwith each other. These polypeptides or proteins can be recombinantlyderived, chemically synthesized or purified from native sources. As usedherein, the term “acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline solution,water and emulsions such as an oil/water or water/oil emulsion, andvarious types of wetting agents. The NAC compositions described hereincan be used, for example, in methods described hereinafter.

[0065] In accordance with another embodiment of the invention,substantially pure nucleic acid molecules, and functional fragmentsthereof, are provided, which encode invention NACs. Exemplary inventionnucleic acid molecules are those comprising substantially the samenucleotide sequence encoding NACO (SEQ ID NO: 1), NACγ (SEQ ID NO: 3),and NACβ (SEQ ID NO: 5).

[0066] The nucleic acid molecules described herein are useful forproducing invention proteins, when such nucleic acids are incorporatedinto a variety of protein expression systems known to those of skill inthe art. In addition, such nucleic acid molecules or fragments thereofcan be labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aninvention NAC gene or mRNA transcript in a given sample. The nucleicacid molecules described herein, and fragments thereof, are also usefulas primers and/or templates in a PCR reaction for amplifying genesencoding invention proteins described herein.

[0067] The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a NAC. One means ofisolating a nucleic acid encoding an NAC polypeptide is to probe amammalian genomic library with a natural or artificially designed DNAprobe using methods well known in the art. DNA probes derived from theNAC gene are particularly useful for this purpose. DNA and cDNAmolecules that encode NAC polypeptides can be used to obtaincomplementary genomic DNA, cDNA or RNA from mammalian (e.g., human,mouse, rat, rabbit, pig, and the like), or other animal sources, or toisolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods described in more detail below. Suchnucleic acids may include, but are not limited to, nucleic acidscomprising substantially the same nucleotide sequence as set forth inSEQ ID NOs:1 (NACβ), 3 (NACγ), and 5 (NACδ).

[0068] Use of the terms “isolated” and/or “purified” and/or“substantially purified” in the present specification and claims as amodifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA,polypeptides or proteins so designated have been produced in such formby the hand of man, and thus are separated from their native in vivocellular environment, and are substantially free of any other species ofnucleic acid or protein. As a result of this human intervention, therecombinant DNAs, RNAs, polypeptides and proteins of the invention areuseful in ways described herein that the DNAs, RNAs, polypeptides orproteins as they naturally occur are not.

[0069] Invention NAC proteins and nucleic acids encoding such, can beobtained from any species of organism, such as prokaryotes, eukaryotes,plants, fungi, vertebrates, invertebrates, and the like. A particularspecies can be mammalian, As used herein, “mammalian” refers to a subsetof species from which an invention NAC is derived, e.g., human, rat,mouse, rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline,and the like. A preferred NAC herein, is human NAC.

[0070] In one embodiment of the present invention, cDNAs encoding theinvention NACs disclosed herein comprise substantially the samenucleotide sequence as the coding region set forth in any of SEQ IDNOs.1, 3 and 5. Preferred cDNA molecules encoding the invention proteinscomprise the same nucleotide sequence as the coding region set forth inany of SEQ ID NOs:1, 3 and 5.

[0071] As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in any of SEQ ID NOs:2, 4, 6, 10 or 12. In anotherembodiment, DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% identity with respectto the reference nucleotide sequence. DNA having at least 70%, morepreferably at least 90%, yet more preferably at least 95%, identity tothe reference nucleotide sequence is preferred.

[0072] This invention also encompasses nucleic acids which differ fromthe nucleic acids shown in SEQ ID NOs:1, 3 and 5, but which have thesame phenotype. Phenotypically similar nucleic acids are also referredto as “functionally equivalent nucleic acids”. As used herein, thephrase “functionally equivalent nucleic acids” encompasses nucleic acidscharacterized by slight and non-consequential sequence variations thatwill function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptides that are thesame as those encoded by the nucleic acids disclosed herein or that haveconservative amino acid variations. For example, conservative variationsinclude substitution of a non-polar residue with another non-polarresidue, or substitution of a charged residue with a similarly chargedresidue. These variations include those recognized by skilled artisansas those that do not substantially alter the tertiary structure of theprotein.

[0073] Further provided are nucleic acids encoding NAC polypeptidesthat, by virtue of the degeneracy of the genetic code, do notnecessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. Preferred nucleic acids encoding the inventionNACs are comprised of nucleotides that encode substantially the sameamino acid sequence as set forth in SEQ ID NOs:2, 4, 6, 10 or 12.

[0074] Thus, an exemplary nucleic acid encoding an invention NAC may beselected from:

[0075] (a) DNA encoding the amino acid sequence set forth in SEQ IDNOs:2, 4, 6, 10 or 12,

[0076] (b) DNA that hybridizes to the DNA of (a) under moderatelystringent conditions, wherein said DNA encodes biologically active NAC,or

[0077] (c) DNA degenerate with respect to (b) wherein said DNA encodesbiologically active NAC.

[0078] Hybridization refers to the binding of complementary strands ofnucleic acid (i.e., sense:antisense strands or probe:target-DNA) to eachother through hydrogen bonds, similar to the bonds that naturally occurin chromosomal DNA. Stringency levels used to hybridize a given probewith target-DNA can be readily varied by those of skill in the art.

[0079] The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

[0080] As used herein, the phrase “moderately stringent hybridization”refers to conditions that permit target-DNA to bind a complementarynucleic acid that has about 60% identity, preferably about 75% identity,more preferably about 85% identity to the target DNA; with greater thanabout 90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5× Denhart's solution, 5× SSPE, 0.2% SDSat 42° C., followed by washing in 0.2× SSPE, 0.2% SDS, at 65° C.

[0081] The phrase “high stringency hybridization” refers to conditionsthat permit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein). High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhart'ssolution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.1× SSPE,and 0.1% SDS at 65° C.

[0082] The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 42° C., followed by washing in 1× SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, (1989)) are well known to those of skill in the art as are othersuitable hybridization buffers.

[0083] As used herein, the term “degenerate” refers to codons thatdiffer in at least one nucleotide from a reference nucleic acid, e.g.,SEQ ID NOs:1, 3 and 5, but encode the same amino acids as the referencenucleic acid. For example, codons specified by the triplets “UCU”,“UCC”, “UCA”, and “UCG” are degenerate with respect to each other sinceall four of these codons encode the amino acid serine.

[0084] Preferred nucleic acids encoding the invention polypeptide(s)hybridize under moderately stringent, preferably high stringency,conditions to substantially the entire sequence, or substantial portions(i.e., typically at least 15-30 nucleotides) of the nucleic acidsequence set forth in SEQ ID NOs:1, 3 and 5.

[0085] The invention nucleic acids can be produced by a variety ofmethods well-known in the art, e.g., the methods described herein,employing PCR amplification using oligonucleotide primers from variousregions of SEQ ID NOs:1, 3 and 5, and the like.

[0086] In accordance with a further embodiment of the present invention,optionally labeled NAC-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleic acid sequences encoding novel NACs. Construction ofsuitable mammalian cDNA libraries, including mammalian cDNA libraries,is well-known in the art. Screening of such a cDNA library is initiallycarried out under low-stringency conditions, which comprise atemperature of less than about 42° C., a formamide concentration of lessthan about 50%, and a moderate to low salt concentration.

[0087] Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5× standard saline citrate (SSC; 20× SSCcontains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially the samenucleotide sequence as SEQ ID NOs:1, 3 and 5 are obtained.

[0088] As used herein, a nucleic acid “probe” is single-stranded DNA orRNA, or analogs thereof, that has a sequence of nucleotides thatincludes at least 15, at least 20, at least 50, at least 100, at least200, at least 300, at least 400, or at least 500 contiguous bases thatare the same as (or the complement of) any contiguous bases set forth inany of SEQ ID NOs:1, 3 and 5. Preferred regions from which to constructprobes include 5′ and/or 3′ coding regions of SEQ ID NOs:1, 3 and 5. Inaddition, the entire cDNA encoding region of an invention NAC, or theentire sequence corresponding to SEQ ID NOs:1, 3 and 5, may be used as aprobe. Probes may be labeled by methods well-known in the art, asdescribed hereinafter, and used in various diagnostic kits.

[0089] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

[0090] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturation to forma fluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

[0091] In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

[0092] Also provided are antisense-nucleic acids having a sequencecapable of binding specifically with full-length or any portion of anmRNA that encodes NAC polypeptides so as to prevent translation of themRNA. The antisense-nucleic acid may have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encoding NACpolypeptides. As used herein, the phrase “binding specifically”encompasses the ability of a nucleic acid sequence to recognize acomplementary nucleic acid sequence and to form double-helical segmentstherewith via the formation of hydrogen bonds between the complementarybase pairs. An example of an antisense-nucleic acid is anantisense-nucleic acid comprising chemical analogs of nucleotides.

[0093] Compositions comprising an amount of the antisense-nucleic acid,described above, effective to reduce expression of NAC polypeptides bypassing through a cell membrane and binding specifically with mRNAencoding NAC polypeptides so as to prevent translation and an acceptablehydrophobic carrier capable of passing through a cell membrane are alsoprovided herein. Suitable hydrophobic carriers are described, forexample, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and thelike. The acceptable hydrophobic carrier capable of passing through cellmembranes may also comprise a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type. The structure may be part of a protein known tobind to a cell-type specific receptor.

[0094] Antisense-nucleic acid compositions are useful to inhibittranslation of mRNA encoding invention polypeptides. Syntheticoligonucleotides, or other antisense chemical structures are designed tobind to mRNA encoding NAC polypeptides and inhibit translation of mRNAand are useful as compositions to inhibit expression of NAC associatedgenes in a tissue sample or in a subject.

[0095] In accordance with another embodiment of the invention, kits areprovided for detecting mutations, duplications, deletions,rearrangements and aneuploidies in NAC genes comprising at least oneinvention probe or antisense nucleotide.

[0096] The present invention provides means to modulate levels ofexpression of NAC polypeptides by employing synthetic antisense-nucleicacid compositions (hereinafter SANC) which inhibit translation of mRNAencoding these polypeptides. Synthetic oligonucleotides, or otherantisense-nucleic acid chemical structures designed to recognize andselectively bind to mRNA, are constructed to be complementary tofull-length or portions of an NAC coding strand, including nucleotidesequences set forth in SEQ ID NOs:1, 3 and 5. The SANC is designed to bestable in the blood stream for administration to a subject by injection,or in laboratory cell culture conditions. The SANC is designed to becapable of passing through the cell membrane in order to enter thecytoplasm of the cell by virtue of physical and chemical properties ofthe SANC which render it capable of passing through cell membranes, forexample, by designing small, hydrophobic SANC chemical structures, or byvirtue of specific transport systems in the cell which recognize andtransport the SANC into the cell. In addition, the SANC can be designedfor administration only to certain selected cell populations bytargeting the SANC to be recognized by specific cellular uptakemechanisms which bind and take up the SANC only within select cellpopulations. In a particular embodiment the SANC is an antisenseoligonucleotide.

[0097] For example, the SANC may be designed to bind to a receptor foundonly in a certain cell type, as discussed supra. The SANC is alsodesigned to recognize and selectively bind to target mRNA sequence,which may correspond to a sequence contained within the sequences shownin SEQ ID NOs:1, 3 and 5. The SANC is designed to inactivate target mRNAsequence by either binding thereto and inducing degradation of the mRNAby, for example, RNase I digestion, or inhibiting translation of mRNAtarget sequence by interfering with the binding oftranslation-regulating factors or ribosomes, or inclusion of otherchemical structures, such as ribozyme sequences or reactive chemicalgroups which either degrade or chemically modify the target mRNA. SANCshave been shown to be capable of such properties when directed againstmRNA targets (see Cohen et al., TIPS, 10:435 (1989) and Weintraub, Sci.American, January (1990), pp.40; both incorporated herein by reference).

[0098] In accordance with yet another embodiment of the presentinvention, there is provided a method for the recombinant production ofinvention NAC by expressing the above-described nucleic acid sequencesin suitable host cells. Recombinant DNA expression systems that aresuitable to produce NAC described herein are well-known in the art. Forexample, the above-described nucleotide sequences can be incorporatedinto vectors for further manipulation. As used herein, vector (orplasmid) refers to discrete elements that are used to introduceheterologous DNA into cells for either expression or replicationthereof.

[0099] Suitable expression vectors are well-known in the art, andinclude vectors capable of expressing DNA operatively linked to aregulatory sequence, such as a promoter region that is capable ofregulating expression of such DNA. Thus, an expression vector refers toa recombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

[0100] Prokaryotic transformation vectors are well-known in the art andinclude pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla,Calif.), and the like. Other suitable vectors and promoters aredisclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989,the disclosure of which is incorporated herein by reference in itsentirety.

[0101] Other suitable vectors for transformation of E. coli cellsinclude the pET expression vectors (Novagen, see U.S. Pat. No.4,952,496), e.g., pET11a, which contains the T7 promoter, T7 terminator,the inducible E. coil lac operator, and the lac repressor gene; and pET12a-c, which contain the T7 promoter, T7 terminator, and the E. coliompT secretion signal. Another suitable vector is the pIN-IIIompA2 (seeDuffaud et al., Meth. in Enzymology, 153:492-507, 1987), which containsthe lpp promoter, the lacUV5 promoter operator, the ompA secretionsignal, and the lac repressor gene.

[0102] In accordance with another embodiment of the present invention,there are provided “recombinant cells” containing the nucleic acidmolecules (i.e., DNA or mRNA) of the present invention. Methods oftransforming suitable host cells, preferably bacterial cells, and morepreferably E. coli cells, as well as methods applicable for culturingsaid cells containing a gene encoding a heterologous protein, aregenerally known in the art. See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989).

[0103] Exemplary methods of transformation include, e.g., transformationemploying plasmids, viral, or bacterial phage vectors, transfection,electroporation, lipofection, and the like. The heterologous DNA canoptionally include sequences which allow for its extrachromosomalmaintenance, or said heterologous DNA can be caused to integrate intothe genome of the host (as an alternative means to ensure stablemaintenance in the host).

[0104] Host organisms contemplated for use in the practice of thepresent invention include those organisms in which recombinantproduction of heterologous proteins has been carried out. Examples ofsuch host organisms include bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha andP. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk⁻ cells),insect cells, and the like. Presently preferred host organisms arebacteria. The most preferred bacteria is E. coli.

[0105] In one embodiment, nucleic acids encoding the invention NAC canbe delivered into mammalian cells, either in vivo or in vitro usingsuitable viral vectors well-known in the art. Suitable retroviralvectors, designed specifically for “gene therapy” methods, aredescribed, for example, in WIPO publications WO 9205266 and WO 9214829,which provide a description of methods for efficiently introducingnucleic acids into human cells. In addition, where it is desirable tolimit or reduce the in vivo expression of the invention NAC, theintroduction of the antisense strand of the invention nucleic acid iscontemplated.

[0106] For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., Proc. Natl. Acad. Sci., USA, 89:6099-6103 (1992); Curielet al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum. Gene Ther.,4:14-24 (1993)) are employed to transduce mammalian cells withheterologous NAC nucleic acid. Any of the plasmid expression vectorsdescribed herein may be employed in a TfAdpl-DNA complex.

[0107] In accordance with yet another embodiment of the presentinvention, there are provided anti-NAC antibodies having specificreactivity with an NAC polypeptides of the present invention. Thepresent invention also provides anti-NACβ, anti-NACγ, anti-NACδ,anti-NACβ-X1, or anti-NACγ/δ-X1 antibodies. It should be recognized thatan antibody of the invention can be specific for an epitope that ispresent only in a particular type of NAC or can be specific for anepitope that is common to more than one type of NAC. For example, ananti-NACδ antibody can be specific for only NACδ or for more than onemember of the NAC family. As used herein, the term “antibody” is used inits broadest sense to include polyclonal and monoclonal antibodies, aswell as polypeptide fragments of antibodies that retain a specificbinding activity for a specific antigen of at least about 1×105 M-1. Oneskilled in the art would know that, for example, anti-NACβ antibodyfragments or anti-NACγ antibody fragments such as Fab, F(ab′)2, Fv andFd fragments can retain specific binding activity for a NACβ or a NACγ,respectively, and, thus, are included within the definition of anantibody. In addition, the term “antibody” as used herein includesnaturally occurring antibodies as well as non-naturally occurringantibodies and fragments of antibodies that retain binding activity.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al., Science 246:1275-1281 (1989), which is incorporated herein byreference.

[0108] Invention antibodies can be produced by methods known in the artusing invention polypeptides, proteins or portions thereof as antigens.For example, polyclonal and monoclonal antibodies can be produced bymethods well known in the art, as described, for example, in Harlow andLane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory(1988)), which is incorporated herein by reference. Inventionpolypeptides can be used as immunogens in generating such antibodies.Alternatively, synthetic peptides can be prepared (using commerciallyavailable synthesizers) and used as immunogens. Amino acid sequences canbe analyzed by methods well known in the art to determine whether theyencode hydrophobic or hydrophilic domains of the correspondingpolypeptide. Altered antibodies such as chimeric, humanized, CDR-graftedor bifunctional antibodies can also be produced by methods well known inthe art. Such antibodies can also be produced by hybridoma, chemicalsynthesis or recombinant methods described, for example, in Sambrook etal., supra., and Harlow and Lane, supra. Both anti-peptide andanti-fusion protein antibodies can be used. (see, for example, Bahouthet al., Trends Pharmacol. Sci. 12:338 (1991); Ausubel et al., CurrentProtocols in Molecular Biology (John Wiley and Sons, NY (1989) which areincorporated herein by reference).

[0109] In the case of monoclonal antibodies specific to NAC, it is alsocontemplated herein that the invention includes hybridomas and any othertype of cell line which produces a monoclonal antibody. Methods ofpreparing hybridomas are described for example, in Sambrook et al.,supra., and Harlow and Lane, supra; and preparation of any non-hybridomacell line producing a monoclonal antibody specific to NAC can be carriedout in accordance with the methods known in the art and methodsdescribed herein for protein expression in cells such as bacterialcells, yeast cells, amphibian cells, mammalian cells, and the like.

[0110] Antibody so produced can be used, inter alia, in diagnosticmethods and systems to detect the level of NAC present in a mammalian,preferably human, body sample, such as tissue or vascular fluid. Suchantibodies can also be used for the immunoaffinity or affinitychromatography purification of the invention NAC. In addition, methodsare contemplated herein for detecting the presence of an invention NACprotein in a tissue or cell, comprising contacting the cell with anantibody that specifically binds to NAC polypeptides, under conditionspermitting binding of the antibody to the NAC polypeptides, detectingthe presence of the antibody bound to the NAC polypeptide, and therebydetecting the presence of invention polypeptides. With respect to thedetection of such polypeptides, the antibodies can be used for in vitrodiagnostic or in vivo imaging methods.

[0111] Immunological procedures useful for in vitro detection of targetNAC polypeptides in a sample include immunoassays that employ adetectable antibody. Such immunoassays include, for example, ELISA,Pandex microfluorimetric assay, agglutination assays, flow cytometry,serum diagnostic assays and immunohistochemical staining procedureswhich are well known in the art. An antibody can be made detectable byvarious means well known in the art. For example, a detectable markercan be directly or indirectly attached to the antibody. Useful markersinclude, for example, radionucleotides, enzymes, fluorogens, chromogensand chemiluminescent labels.

[0112] Invention anti-NAC antibodies are contemplated for use herein tomodulate the activity of the NAC polypeptide in living animals, inhumans, or in biological tissues or fluids isolated therefrom. The term“modulate” refers to a compound's ability to increase (e.g., via anagonist) or inhibit (e.g., via an antagonist) the biological activity ofan invention NAC protein, such as the capablity of bindingCARD-containing proteins, NB-ARC-containing proteins, to modulate theactivity of proteases such as caspases, to modulate the activity ofNF-κB, and to modulate apoptosis. Accordingly, compositions comprising acarrier and an amount of an antibody having specificity for NACpolypeptides effective to inhibit naturally occurring ligands or NAPsfrom binding to invention NAC polypeptides are contemplated herein. Forexample, a monoclonal antibody directed to an epitope of an inventionNAC polypeptide including an amino acid sequence set forth in SEQ IDNOs:2, 4, 6, 10 or 12, can be useful for this purpose.

[0113] The present invention further provides transgenic non-humanmammals that are capable of expressing exogenous nucleic acids encodingNAC polypeptides. As employed herein, the phrase “exogenous nucleicacid” refers to nucleic acid sequence which is not native to the host,or which is present in the host in other than its native environment(e.g., as part of a genetically engineered DNA construct). In additionto naturally occurring levels of NAC, invention NAC can either beoverexpressed or underexpressed (such as in the well-known knock-outtransgenics) in transgenic mammals.

[0114] Also provided are transgenic non-human mammals capable ofexpressing nucleic acids encoding NAC polypeptides so mutated as to beincapable of normal activity, i.e., do not express native NAC. Thepresent invention also provides transgenic non-human mammals having agenome comprising antisense nucleic acids complementary to nucleic acidsencoding NAC polypeptides, placed so as to be transcribed into antisensemRNA complementary to mRNA encoding NAC polypeptides, which hybridizesto the mRNA and, thereby, reduces the translation thereof. The nucleicacid may additionally comprise an inducible promoter and/or tissuespecific regulatory elements, so that expression can be induced, orrestricted to specific cell types. Examples of nucleic acids are DNA orcDNA having a coding sequence substantially the same as the codingsequence shown in SEQ ID NOs:1, 3 or 5. An example of a non-humantransgenic mammal is a transgenic mouse. Examples of tissuespecificity-determining elements are the metallothionein promoter andthe L7 promoter.

[0115] Animal model systems which elucidate the physiological andbehavioral roles of NAC polypeptides are also provided, and are producedby creating transgenic animals in which the expression of the NACpolypeptide is altered using a variety of techniques. Examples of suchtechniques include the insertion of normal or mutant versions of nucleicacids encoding an NAC polypeptide by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal. (See, forexample, Hogan et al., Manipulating the Mouse Embryo: A LaboratoryManual (Cold Spring Harbor Laboratory, (1986)).

[0116] Also contemplated herein, is the use of homologous recombinationof mutant or normal versions of NAC genes with the native gene locus intransgenic animals, to alter the regulation of expression or thestructure of NAC polypeptides (see, Capecchi et al., Science 244:1288(1989); Zimmer et al., Nature 338:150 (1989); which are incorporatedherein by reference). Homologous recombination techniques are well knownin the art. Homologous recombination replaces the native (endogenous)gene with a recombinant or mutated gene to produce an animal that cannotexpress native (endogenous) protein but can express, for example, amutated protein which results in altered expression of NAC polypeptides.

[0117] In contrast to homologous recombination, microinjection addsgenes to the host genome, without removing host genes. Microinjectioncan produce a transgenic animal that is capable of expressing bothendogenous and exogenous NAC. Inducible promoters can be linked to thecoding region of nucleic acids to provide a means to regulate expressionof the transgene. Tissue specific regulatory elements can be linked tothe coding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of specific ligands, i.e., agonists andantagonists, which activate or inhibit NAC protein responses.

[0118] A further embodiment of the invention provides a method toidentify agents that can effectively alter NAC activity, for example theability of NAC to association with one or more heterologous proteins.Thus, the present invention provides a screening assay useful foridentifying an effective agent, which can alter the association of a NACwith a NAC associated protein, such as a CARD-containing protein and/oran NB-ARC-containing protein. Since CARD-containing proteins andNB-ARC-containing proteins are involved in apoptosis, the identificationof such effective agents can be useful for modulating the level ofapoptosis in a cell in a subject having a pathology characterized by anincreased or decreased level of apoptosis.

[0119] Further, since invention NAC proteins comprise CARD domains,effective agents can be useful for modulation of any other CARD domainactivity. These additional CARD domain activities include, for example,NF-κB activity modulation, cytokine receptor signal transduction, andcaspase activation/inhibition, regardless of whether the effectedcaspase is involved in apoptosis or some alternative cellular processsuch as proteolytic processing and activation of inflammatory cytokines.

[0120] As used herein, the term “agent” means a chemical or biologicalmolecule such as a simple or complex organic molecule, a peptide, apeptido-mimetic, a protein or an oligonucleotide that has the potentialfor altering the association of NAC with a heterologous protein oraltering the ability of NAC to self-associate or altering the nucleotidebinding and/or hydrolysis activity of NAC. In addition, the term“effective agent” is used herein to mean an agent that can, in fact,alter the association of NAC with a heterologous protein or altering theability of NAC to self-associate or altering the nucleotide bindingand/or hydrolysis activity of NAC. For example, an effective agent maybe an anti-NAC antibody or a NAC-associated-protein.

[0121] As used herein, the term “alter the association” means that theassociation between two specifically interacting proteins either isincreased or is decreased due to the presence of an effective agent. Asa result of an altered association of NAC with another protein in acell, the activity of the NAC or the NAC associated protein can beincreased or decreased, thereby modulating a biological process, forexample, the level of apoptosis in the cell. As used herein, the term“alter the activity” means that the agent can increase or decrease theactivity of a NAC in a cell, thereby modulating a biological process ina cell, for example, the level of apoptosis in the cell. For example, aneffective agent can increase or decrease the NB-ARC:NB-ARC-associatingactivity of a NAC, without affecting the association of the NAC with aCARD-containing protein. Modulation of the ATP hydrolysis activity canmodulate the ability of NAC proteins to associate with otherNB-ARC-containing proteins, such as Apaf-1, thereby modulating anyprocess effected by such association between NAC and anNB-ARC-containing protein. Similarly, the term “alters the association”of NAC with another protein refers to increasing or decreasing, orotherwise changing the association between a NAC and a protein thatspecifically binds to NAC (i.e., a NAC associated protein).

[0122] An effective agent can act by interfering with the ability of aNAC to associate with another protein, or can act by causing thedissociation of NAC from a complex with a NAC-associated protein,wherein the ratio of bound NAC to free NAC is related to the level of abiological process, for example, apoptosis, in a cell. For example,binding of a ligand to a NAC-associated protein can allow theNAC-associated protein, in turn, to bind a NAC. The association, forexample, of a CARD-containing protein and a NAC can result in activationor inhibition of the NB-ARC:NB-ARC-associating activity of NAC. In thepresence of an effective agent, the association of a NAC and aCARD-containing protein can be altered, which can thereby alter theactivation of caspases in the cell. As a result of the altered caspaseactivation, the level of apoptosis in a cell can be increased ordecreased. Thus, the identification of an effective agent that altersthe association of NAC with another protein can allow for the use of theeffective agent to increase or decrease the level of apoptosis in acell.

[0123] An effective agent can be useful, for example, to increase thelevel of apoptosis in a cell such as a cancer cell, which ischaracterized by having a decreased level of apoptosis as compared toits normal cell counterpart. An effective agent also can be useful, forexample, to decrease the level of apoptosis in a cell such as a Tlymphocyte in a subject having a viral disease such as acquiredimmunodeficiency syndrome, which is characterized by an increased levelof apoptosis in an infected T cell as compared to a normal T cell. Thus,an effective agent can be useful as a medicament for altering the levelof apoptosis in a subject having a pathology characterized by increasedor decreased apoptosis. In addition, an effective agent can be used, forexample, to decrease the level of apoptosis and, therefore, increase thesurvival time of a cell such as a hybridoma cell in culture. The use ofan effective agent to prolong the survival of a cell in vitro cansignificantly improve bioproduction yields in industrial tissue cultureapplications.

[0124] A NAC that lacks the ability to bind the NB-ARC domain of anotherprotein but retains the ability to self-associate via its CARD domain orto bind to other CARD-containing proteins is an example of an effectiveagent, since the expression of a non-NB-ARC-associating NAC in a cellcan alter the association of a the endogenous NAC protein with itself orwith NAC associated proteins.

[0125] Thus, it should be recognized that a mutation of a NAC can be aneffective agent, depending, for example, on the normal level ofNAC/NAC-associated protein that occurs in a particular cell type. Inaddition, an active fragment of a NAC can be an effective agent,provided the active fragment can alter the association of NAC andanother protein in a cell. Such active fragments, which can be peptidesas small as about five amino acids, can be identified, for example, byscreening a peptide library (see, for example, Ladner et al., U.S. Pat.No. 5,223,409, which is incorporated herein by reference) to identifypeptides that can bind a NAC-associated protein.

[0126] Similarly, a peptide or polypeptide portion of a NAC-associatedprotein also can be an effective agent. A peptide such as the C-terminalpeptide of NAC-associated protein can be useful, for example, fordecreasing the association of NAC with a CARD-containing protein or aNB-ARC-containing protein in a cell by competing for binding to the NAC.A non-naturally occurring peptido-mimetic also can be useful as aneffective agent. Such a peptido-mimetic can include, for example, apeptoid, which is peptide-like sequence containing N-substitutedglycines, or an oligocarbamate. A peptido-mimetic can be particularlyuseful as an effective agent due, for example, to having an increasedstability to enzymatic degradation in vivo.

[0127] A screening assay to identify an effective agent can be performedin vivo using the two hybrid system or can be performed in vitro asdisclosed herein. The yeast two hybrid system, for example, can be usedto screen a panel of agents to identify effective agents that alter theassociation of NAC with another protein. An effective agent can beidentified by detecting an altered level of transcription of a reportergene. For example, the level of transcription of a reporter gene due tothe bridging of a DNA-binding domain and trans-activation domain by aNAP and NAC hybrids can be determined in the absence and in the presenceof an agent. An effective agent, which alters the association betweenNAC and another protein, can be identified by a proportionately alteredlevel of transcription of the reporter gene as compared to the controllevel of transcription in the absence of the agent.

[0128] As understood by those of skill in the art, assay methods foridentifying agents that modulate NAC activity generally requirecomparison to a control. For example, one type of a “control” is a cellor culture that is treated substantially the same as the test cell ortest culture exposed to the agent, with the distinction that the“control” cell or culture is not exposed to the agent. Another type of“control” cell or culture may be a cell or culture that is identical tothe transfected cells, with the exception that the “control” cell orculture do not express native proteins. Accordingly, the response of thetransfected cell to agent is compared to the response (or lack thereof)of the “control” cell or culture to the same agent under the samereaction conditions. Similarly, a “control” can be the extract,partially purified or not, of a cell not exposed to the agent or notexpressing certain native proteins. A “control” may also be an isolatedcompound, for example, a protein (e.g., Skp-1 as used in Examples),which is known to not specifically associate with NAC proteins.

[0129] Accordingly, in accordance with another embodiment of the presentinvention, there is provided a method of identifying an effective agentthat alters the association of a NB-ARC and CARD-containing protein(NAC) with a NAC associated protein (NAP), by the steps of:

[0130] a. contacting said NAC and NAP proteins, under conditions thatallow the NAC and NAP proteins to associate, with an agent suspected ofbeing able to alter the association of the NAC and NAP proteins; and

[0131] b. detecting the altered association of the NAC and NAP proteins,wherein the altered association identifies an effective agent.

[0132] Methods well-known in the art for detecting the alteredassociation of the NAC and NAP proteins, for example, measuringprotein:protein binding, protein degradation or apoptotic activity canbe employed in bioassays described herein to identify agents as agonistsor antagonists of NAC proteins. As described herein, NAC proteins havethe ability to self-associate. Thus, methods for identifying effectiveagents that alter the association of a NAC protein NAP will also beuseful for identifying effective agents that alter the ability of NAC toself-associate. Similarly, CARD-X proteins have the ability to interactwith other CARD-containing proteins and to self-associate. Thus, methodsfor identifying effective agents that alter the association of a NAC andanother protein will also be useful for identifying effective agentsthat alter the ability of CARD-X to self-associate or to associate witha heterologous CARD-containing protein.

[0133] As used herein, “conditions that allow said NAC and NAP proteinsto associate” refers to environmental conditions in which NAC:NAPspecifically associate. Such conditions will typically be aqueousconditions, with a pH between 3.0 and 11.0, and temperature below 100°C. Preferably, the conditions will be aqueous conditions with saltconcentrations below the equivalent of 1 M NaCl, and pH between 5.0 and9.0, and temperatures between 0° C. and 50° C. Most preferably, theconditions will range from physiological conditions of normal yeast ormammalian cells, or conditions favorable for carrying out in vitroassays such as immunoprecipitation and GST-NAC:NAP association assays,and the like.

[0134] In yet another embodiment of the present invention, there areprovided methods for modulating the caspase modulating activity mediatedby NAC proteins, the method comprising:

[0135] contacting an NAC protein with an effective, modulating amount ofan agonist or antagonist identified by the above-described bioassays.

[0136] The present invention also provides in vitro screening assays.Such screening assays are particularly useful in that they can beautomated, which allows for high through-put screening, for example, ofrandomly or rationally designed agents such as drugs, peptidomimetics orpeptides in order to identify those agents that effectively alter theassociation of NAC and NAP proteins or the activity of a NAC and,thereby, modulate apoptosis. An in vitro screening assay can utilize,for example, a NAC or a NAC fusion protein such as aNAC-glutathione-S-transferase fusion protein (GST/NAC; see Examples).For use in the in vitro screening assay, the NAC or NAC fusion proteinshould have an affinity for a solid substrate as well as the ability toassociate with a NAC-associated protein. For example, when a NAC is usedin the assay, the solid substrate can contain a covalently attachedanti-NAC antibody. Alternatively, a GST/NAC fusion protein can be usedin the assay and the solid substrate can contain covalently attachedglutathione, which is bound by the GST component of the GST/NAC fusionprotein. Similarly, a NAC-associated protein, or a GST/CARD-containingprotein or GST/NB-ARC-containing protein fusion protein can be used inan in vitro assay as described herein.

[0137] An in vitro screening assay can be performed by allowing a NAC orNAC-fusion protein, for example, to bind to the solid support, thenadding a NAC-associated protein and an agent to be tested. Controlreactions, which do not contain an agent, can be performed in parallel.Following incubation under suitable conditions, which include, forexample, an appropriate buffer concentration and pH and time andtemperature that permit binding of the particular NAC and NAC-associatedprotein, the amount of protein that has associated in the absence of anagent and in the presence of an agent can be determined. The associationof a NAC-associated protein with a NAC protein can be detected, forexample, by attaching a detectable moiety such as a radionuclide or afluorescent label to a NAC-associated protein and measuring the amountof label that is associated with the solid support, wherein the amountof label detected indicates the amount of association of theNAC-associated protein with a NAC protein. An effective agent isdetermined by comparing the amount of specific binding in the presenceof an agent as compared to the control level of binding, wherein aneffective agent alters the association of NAC with the NAC-assocatedprotein. Such an assay is particularly useful for screening a panel ofagents such as a peptide library in order to detect an effective agent.

[0138] The invention further provides methods for introducing a nucleicacid encoding a NAC into a cell in a subject, for example, for genetherapy. Viruses are specialized infectious agents that can elude hostdefense mechanisms and can infect and propagate in specific cell types.Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing invention nucleic acidencoding an NAC protein into mammalian cells (e.g., vascular tissuesegments) are well known in the art. These viral vectors include, forexample, Herpes simplex virus vectors (e.g., Geller et al., Science,241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al.,Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirus vectors(Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.78-84), Moloney murine leukemia virus vectors (Danos et al., Proc. Natl.Acad. Sci., USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan etal., Proc. Natl. Acad. Sci., USA, 81:3655-3659 (1984); Jones et al.,Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626 (1988);Cotten et al., Proc. Natl. Acad. Sci., USA, 89:6094-6098 (1992); Grahamet al., Meth. Mol. Biol., 7:109-127 (1991)), adeno-associated virusvectors, retrovirus vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and4,650,764), and the like. Especially preferred viral vectors are theadenovirus and retroviral vectors.

[0139] Suitable retroviral vectors for use herein are described, forexample, in U.S. Pat. No. 5,252,479, and in WIPO publications WO92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829,incorporated herein by reference, which provide a description of methodsfor efficiently introducing nucleic acids into human cells using suchretroviral vectors. Other retroviral vectors include, for example, themouse mammary tumor virus vectors (e.g., Shackleford et al., Proc. Natl.Acad. Sci. USA, 85:9655-9659 (1988)), and the like.

[0140] In particular, the specificity of viral vectors for particularcell types can be utilized to target predetermined cell types. Thus, theselection of a viral vector will depend, in part, on the cell type to betargeted. For example, if a neurodegenerative disease is to be treatedby increasing the level of a NAC in neuronal cells affected by thedisease, then a viral vector that targets neuronal cells can be used. Avector derived from a herpes simplex virus is an example of a viralvector that targets neuronal cells (Battleman et al., J. Neurosci.13:941-951 (1993), which is incorporated herein by reference).Similarly, if a disease or pathological condition of the hematopoieticsystem is to be treated, then a viral vector that is specific for aparticular blood cell or its precursor cell can be used. A vector basedon a human immunodeficiency virus is an example of such a viral vector(Carroll et al., J. Cell. Biochem. 17E:241 (1993), which is incorporatedherein by reference). In addition, a viral vector or other vector can beconstructed to express a nucleic acid encoding a NAC in a tissuespecific manner by incorporating a tissue-specific promoter or enhancerinto the vector (Dai et al., Proc. Natl. Acad. Sci. USA 89:10892-10895(1992), which is incorporated herein by reference).

[0141] For gene therapy, a vector containing a nucleic acid encoding aNAC or an antisense nucleotide sequence can be administered to a subjectby various methods. For example, if viral vectors are used,administration can take advantage of the target specificity of thevectors. In such cases, there in no need to administer the vectorlocally at the diseased site. However, local administration can be aparticularly effective method of administering a nucleic acid encoding aNAC. In addition, administration can be via intravenous or subcutaneousinjection into the subject. Following injection, the viral vectors willcirculate until they recognize host cells with the appropriate targetspecificity for infection. Injection of viral vectors into the spinalfluid also can be an effective mode of administration, for example, intreating a neurodegenerative disease.

[0142] Receptor-mediated DNA delivery approaches also can be used todeliver a nucleic acid molecule encoding a NAC into cells in atissue-specific manner using a tissue-specific ligand or an antibodythat is non-covalently complexed with the nucleic acid molecule via abridging molecule (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wuand Wu, J. Biol. Chem. 262:4429-4432 (1987), each of which isincorporated herein by reference). Direct injection of a naked or anucleic acid molecule encapsulated, for example, in cationic liposomesalso can be used for stable gene transfer into non-dividing or dividingcells in vivo (Ulmer et al., Science 259:1745-1748 (1993), which isincorporated herein by reference). In addition, a nucleic acid moleculeencoding a NAC can be transferred into a variety of tissues using theparticle bombardment method (Williams et al., Proc. Natl. Acad. Sci. USA88:2726-2730 (1991), which is incorporated herein by reference). Suchnucleic acid molecules can be linked to the appropriate nucleotidesequences required for transcription and translation.

[0143] A particularly useful mode of administration of a nucleic acidencoding a NAC is by direct inoculation locally at the site of thedisease or pathological condition. Local administration can beadvantageous because there is no dilution effect and, therefore, thelikelihood that a majority of the targeted cells will be contacted withthe nucleic acid molecule is increased. Thus, local inoculation canalleviate the targeting requirement necessary with other forms ofadministration and, if desired, a vector that infects all cell types inthe inoculated area can be used. If expression is desired in only aspecific subset of cells within the inoculated area, then a promotor, anenhancer or other expression element specific for the desired subset ofcells can be linked to the nucleic acid molecule. Vectors containingsuch nucleic acid molecules and regulatory elements can be viralvectors, viral genomes, plasmids, phagemids and the like. Transfectionvehicles such as liposomes also can be used to introduce a non-viralvector into recipient cells. Such vehicles are well known in the art.

[0144] The present invention also provides therapeutic compositionsuseful for practicing the therapeutic methods described herein.Therapeutic compositions of the present invention, such aspharmaceutical compositions, contain a physiologically compatiblecarrier together with an invention NAC (or functional fragment thereof),a NAC modulating agent, such as a compound (agonist or antagonist)identified by the methods described herein, or an anti-NAC antibody, asdescribed herein, dissolved or dispersed therein as an activeingredient. In a preferred embodiment, the therapeutic composition isnot immunogenic when administered to a mammal or human patient fortherapeutic purposes.

[0145] As used herein, the terms “pharmaceutically acceptable”,“physiologically compatible” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

[0146] The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well known in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions; however, solid forms suitable forsolution, or suspension, in liquid prior to use can also be prepared.The preparation can also be emulsified.

[0147] The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

[0148] The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

[0149] Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

[0150] Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

[0151] Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary additional liquid phasesinclude glycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

[0152] As described herein, an “effective amount” is a predeterminedamount calculated to achieve the desired therapeutic effect, e.g., tomodulate the protein degradation activity of an invention NAC protein.The required dosage will vary with the particular treatment and with theduration of desired treatment; however, it is anticipated that dosagesbetween about 10 micrograms and about 1 milligram per kilogram of bodyweight per day will be used for therapeutic treatment. It may beparticularly advantageous to administer such compounds in depot orlong-lasting form as discussed hereinafter. A therapeutically effectiveamount is typically an amount of an NAC-modulating agent or compoundidentified herein that, when administered in a physiologicallyacceptable composition, is sufficient to achieve a plasma concentrationof from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml andusually 5 to 10 μg/ml. Therapeutic invention anti-NAC antibodies can beadministered in proportionately appropriate amounts in accordance withknown practices in this art.

[0153] Also provided herein are methods of treating pathologies, saidmethod comprising administering an effective amount of an inventiontherapeutic composition. Such compositions are typically administered ina physiologically compatible composition.

[0154] Exemplary diseases related to abnormal cell proliferationcontemplated herein for treatment according to the present inventioninclude cancer pathologies, keratinocyte hyperplasia, neoplasia, keloid,benign prostatic hypertrophy, inflammatory hyperplasia, fibrosis, smoothmuscle cell proliferation in arteries following balloon angioplasty(restenosis), and the like. Exemplary cancer pathologies contemplatedherein for treatment include, gliomas, carcinomas, adenocarcinomas,sarcomas, melanomas, hamartomas, leukemias, lymphomas, and the like.

[0155] Methods of treating pathologies of abnormal cell proliferationwill include methods of modulating the activity of one or more oncogenicproteins, wherein the oncogenic proteins specifically interact with NAC.Methods of modulating the activity of such oncogenic proteins willinclude contacting the oncogenic protein with a substantially pure NACor an active fragment (i.e., oncogenic protein-binding fragment)thereof. This contacting will modulate the activity of the oncogenicprotein, thereby providing a method of treating a pathology caused bythe oncogenic protein. Further methods of modulating the activity ofoncogenic proteins will include contacting the oncogenic protein with anagent, wherein the agent modulates the interactions between NAC and theoncogenic protein.

[0156] Also contemplated herein, are therapeutic methods using inventionpharmaceutical compositions for the treatment of pathological disordersin which there is too little cell division, such as, for example, bonemarrow aplasias, immunodeficiencies due to a decreased number oflymphocytes, and the like. Methods of treating a variety of inflammatorydiseases with invention therapeutic compositions are also contemplatedherein, such as treatment of sepsis, fibrosis (e.g., scarring),arthritis, graft versus host disease, and the like.

[0157] The present invention also provides methods for diagnosing apathology that is characterized by an increased or decreased level ofapoptosis in a cell to determine whether the increased or decreasedlevel of apoptosis is due, for example, to increased or decreasedexpression of a NAC in the cell or to expression of a variant NAC. Theidentification of such a pathology, which can be due to alteredassociation of a NAC with a NAC-associated protein in a cell, can allowfor intervention therapy using an effective agent or a nucleic acidmolecule or an antisense nucleotide sequence as described above. Ingeneral, a test sample can be obtained from a subject having a pathologycharacterized by having or suspected of having increased or decreasedapoptosis and can be compared to a control sample from a normal subjectto determine whether a cell in the test sample has, for example,increased or decreased expression of NAC. The level of a NAC in a cellcan be determined by contacting a sample with a reagent such as ananti-NAC antibody or a NAC-associated protein, either of which canspecifically bind a NAC. For example, the level of a NAC in a cell candetermined by well known immunoassay or immunohistochemical methodsusing an anti-NAC antibody (see, for example, Reed et al., supra, 1992;see, also, Harlow and Lane, supra, (1988)). As used herein, the term“reagent” means a chemical or biological molecule that can specificallybind to a NAC or to a bound NAC/NAC-associated protein complex. Forexample, either an anti-NAC antibody or a NAC-associated protein can bea reagent for a NAC, whereas either an anti-NAC antibody or ananti-NAC-associated protein antibody can be a reagent for aNAC/NAC-associated protein complex.

[0158] As used herein, the term “test sample” means a cell or tissuespecimen that is obtained from a subject and is to be examined forexpression of a NAC in a cell in the sample. A test sample can beobtained, for example, during surgery or by needle biopsy and can beexamined using the methods described herein to diagnose a pathologycharacterized by increased or decreased apoptosis. Increased ordecreased expression of a NAC in a cell in a test sample can bedetermined by comparison to an expected normal level for a NAC in aparticular cell type. A normal range of NAC levels in various cell typescan be determined by sampling a statistically significant number ofnormal subjects. In addition, a control sample can be evaluated inparallel with a test sample in order to determine whether a pathologycharacterized by increased or decreased apoptosis is due to increased ordecreased expression of a NAC. The test sample can be examined using,for example, immunohistochemical methods as described above or thesample can be further processed and examined. For example, an extract ofa test sample can be prepared and examined to determine whether a NACthat is expressed in a cell in the sample can associate with aNAC-associated protein in the same manner as a NAC from a control cellor whether, instead, a variant NAC is expressed in the cell.

[0159] In accordance with another embodiment of the present invention,there are provided diagnostic systems, preferably in kit form,comprising at least one invention nucleic acid encoding NAC, NACprotein, and/or anti-NAC antibody described herein, in a suitablepackaging material. In one embodiment, for example, the diagnosticnucleic acids are derived from any of SEQ ID NOs:1, 3 and 5. Inventiondiagnostic systems are useful for assaying for the presence or absenceof nucleic acid encoding NAC in either genomic DNA or in transcribednucleic acid (such as mRNA or cDNA) encoding NAC.

[0160] A suitable diagnostic system includes at least one invention NACnucleic acid, NAC protein, and/or anti-NAC antibody, preferably two ormore invention nucleic acids, proteins and/or antibodies, as aseparately packaged chemical reagent(s) in an amount sufficient for atleast one assay. Instructions for use of the packaged reagent are alsotypically included. Those of skill in the art can readily incorporateinvention nucleic probes and/or primers into kit form in combinationwith appropriate buffers and solutions for the practice of the inventionmethods as described herein.

[0161] As employed herein, the phrase “packaging material” refers to oneor more physical structures used to house the contents of the kit, suchas invention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular sequence encoding NAC including the nucleotidesequences set forth in SEQ ID NOs:1, 3 and 5 or mutations or deletionstherein, thereby diagnosing the presence of, or a predisposition for,cancer. In addition, the packaging material contains instructionsindicating how the materials within the kit are employed both to detecta particular sequence and diagnose the presence of, or a predispositionfor, cancer.

[0162] The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed.

[0163] “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

[0164] A diagnostic assay should include a simple method for detectingthe amount of a NAC in a sample that is bound to the reagent. Detectioncan be performed by labeling the reagent and detecting the presence ofthe label using well known methods (see, for example, Harlow and Lane,supra, 1988; chap. 9, for labeling an antibody). A reagent can belabeled with various detectable moieties including a radiolabel, anenzyme, biotin or a fluorochrome. Materials for labeling the reagent canbe included in the diagnostic kit or can be purchased separately from acommercial source. Following contact of a labeled reagent with a testsample and, if desired, a control sample, specifically bound reagent canbe identified by detecting the particular moiety.

[0165] A labeled antibody that can specifically bind the reagent alsocan be used to identify specific binding of an unlabeled reagent. Forexample, if the reagent is an anti-NAC antibody, a second antibody canbe used to detect specific binding of the anti-NAC antibody. A secondantibody generally will be specific for the particular class of thefirst antibody. For example, if an anti-NAC antibody is of the IgGclass, a second antibody will be an anti-IgG antibody. Such secondantibodies are readily available from commercial sources. The secondantibody can be labeled using a detectable moiety as described above.When a sample is labeled using a second antibody, the sample is firstcontacted with a first antibody, then the sample is contacted with thelabeled second antibody, which specifically binds to the first antibodyand results in a labeled sample.

[0166] In accordance with another embodiment of the invention, a methodis provided to identify NAC-associated proteins. As used herein, theterm “NAC-associated protein” or “NAP” means a protein that canspecifically bind to NAC or its alternative isoforms. Because NACproteins are known to self-associate, NAC proteins are encompassed bythe term NAP. An exemplary NAP is a protein or a polypeptide portion ofa protein that can bind the NB-ARC, CARD, LRR, or TIM-Barrel-likedomains of NAC. Similarly, the term “CARD-X Associated Protein” or “CAP”refers to a protein that can bind specifically to the CARD-X protein.Likewise, since CARD-X proteins are known to self-associate, CARD-Xproteins are encompassed by the term CAP. A NAP or CAP can beidentified, for example, using in vitro protein binding assays similarto those described in the Examples, by Yeast Two-Hybrid assays similarto those described in the Examples, or by other types ofprotein-interaction assays and methods.

[0167] Using NAC or CARD-X, it is clear to one skilled in the art ofprotein purification, protein interaction cloning, or proteinmass-spectrometry, that NAPs or CAPs can be identified using the methodsdisclosed herein.

[0168] Although the term “NAP” or “CAP” is used generally, it should berecognized that a NAP or CAP that is identified using an assay describedherein can be a portion of a protein, which is considered to be acandidate NAP or CAP. As used herein, the term “active fragment” of aNAP or CAP refers to a protein that corresponds to a polypeptidesequence that can bind NAC or CARD-X, respectively, but that consists ofonly a portion of the full length protein. Although such polypeptidesare considered NAPs or CAPs, it is well known that a cDNA sequenceobtained from a cDNA library may not encode the full length protein.Thus, a cDNA can encode a polypeptide that is only a portion of a fulllength protein but, nevertheless, assumes an appropriate conformationand contains a sufficient region so as to bind NAC or CARD-X. However,in the full length protein, the polypeptide can assume a conformationthat does not bind NAC or CARD-X, due for example to steric blocking ofthe NAP or CAP binding site. Such a full length protein is also anexample of a NAP or CAP, wherein NAC-binding or CARD-X-binding activitycan be activated under the appropriate conditions (i.e.,phosphorylation, proteolysis, protein binding, pH change, and the like).For convenience of discussion, the terms “NAP” and “CAP”, as usedherein, are intended to include a NAP or CAP, respectively, and activefragments thereof.

[0169] Since CARD-containing proteins are commonly involved inapoptosis, the association of a NAP or CAP with NAC or CARD-X can affectthe level of apoptosis in a cell. The identification by use of themethods described herein of various NAPs or CAPs can provide thenecessary insight into cell death or signal transduction pathwayscontrolled by NAC or CARD-X, allowing for the development of assays thatare useful for identifying agents that effectively alter the associationof a NAP with NAC or a CAP with CARD-X. Such agents can be useful, forexample, for providing effective therapy for a cancer in a subject orfor treating an autoimmune disease. These same assays can be used foridentification of agents that modulate the self-association of NAC viaits CARD domain, NB-ARC domain, or other domains within this protein;and, they can be used for identification of agents that modulate theself-association of CARD-X with itself via its CARD domain or otherdomains found within this protein.

[0170] In a normal cell, a steady state level of association of NAP andNAC proteins likely occurs. This steady state level of association ofNAP and NAC proteins in a particular cell type can determine the normallevel of apoptosis in that cell type. An increase or decrease in thesteady state level of association of NAP and NAC proteins in a cell canresult in an increased or decreased level of apoptosis in the cell,which can result in a pathology in a subject. The normal association ofNAP and NAC proteins in a cell can be altered due, for example, to theexpression in the cell of a variant NAP or NAC protein, respectively,either of which can compete with the normal binding function of NAC and,therefore, can decrease the association of NAP and NAC proteins in acell. The term “variant” is used generally herein to mean a protein thatis different from the NAP or NAC protein that normally is found in aparticular cell type. In addition, the normal association of NAP and NACproteins in a cell can be increased or decreased due, for example, tocontact of the cell with an agent such as a drug that can effectivelyalter the association of NAP and NAC proteins in a cell.

[0171] NB-ARC and CARD domain proteins of the invention, NACβ, NACγ andNACδ, were characterized, for example, using an in vitro binding assayand CARD-containing proteins were further characterized using the yeasttwo hybrid system. An in vivo transcription activation assay such as theyeast two hybrid system is particularly useful for identifying andmanipulating the association of proteins. In addition, the resultsobserved in such an assay likely mirror the events that naturally occurin a cell. Thus, the results obtained in such an in vivo assay can bepredictive of results that can occur in a cell in a subject such as ahuman subject.

[0172] A transcription activation assay such as the yeast two hybridsystem is based on the modular nature of transcription factors, whichconsist of functionally separable DNA-binding and trans-activationdomains. When expressed as separate proteins, these two domains fail tomediate gene transcription. However, transcription activation activitycan be restored if the DNA-binding domain and the trans-activationdomain are bridged together due, for example, to the association of twoproteins. The DNA-binding domain and trans-activation domain can bebridged, for example, by expressing the DNA-binding domain andtrans-activation domain as fusion proteins (hybrids), provided that theproteins that are fused to the domains can associate with each other.The non-covalent bridging of the two hybrids brings the DNA-binding andtrans-activation domains together and creates a transcriptionallycompetent complex. The association of the proteins is determined byobserving transcriptional activation of a reporter gene (see Example I).

[0173] The yeast two hybrid systems exemplified herein use variousstrains of S. cerevisiae as host cells for vectors that express thehybrid proteins. A transcription activation assay also can be performedusing, for example, mammalian cells. However, the yeast two hybridsystem is particularly useful due to the ease of working with yeast andthe speed with which the assay can be performed. For example, yeast hostcells containing a lacZ reporter gene linked to a LexA operator sequencewere used to demonstrate that the CARD_(L) domain of NAC (amino acidresidues 1128-1473 of SEQ ID NO:2) can interact with severalCARD-containing proteins (see Examples). For example, in one case theDNA-binding domain consisted of the LexA DNA-binding domain, which bindsthe LexA promoter, fused to the CARD_(L) domain of NAC and thetrans-activation domain consisted of the B42 acidic region separatelyfused to several cDNA sequences which encoded CARD-containing proteins.When the LexA domain was non-covalently bridged to a trans-activationdomain fused to a CARD-containing protein, the association activatedtranscription of the reporter gene.

[0174] A NAP, for example, a CARD-containing protein or anNB-ARC-containing protein also can be identified using an in vitro assaysuch as an assay utilizing, for example, a glutathione-S-transferase(GST) fusion protein as described in the Examples. Such an in vitroassay provides a simple, rapid and inexpensive method for identifyingand isolating a NAP. Such an in vitro assay is particularly useful inconfirming results obtained in vivo and can be used to characterizespecific binding domains of a NAP. For example, a GST/CARD_(L) fusionprotein can be expressed and can be purified by binding to an affinitymatrix containing immobilized glutathione. If desired, a sample that cancontains a CARD-containing protein or active fragments of aCARD-containing protein can be passed over an affinity column containingbound GST/CARD_(L) and a CARD-containing protein that binds to CARD_(L)can be obtained. In addition, GST/CARD_(L) can be used to screen a cDNAexpression library, wherein binding of the GST/CARD_(L) fusion proteinto a clone indicates that the clone contains a cDNA encoding aCARD-containing protein.

[0175] In another embodiment of the invention, methods are provided formonitoring the progress of treatment for a pathology that ischaracterized by an increased or decreased level of apoptosis in a cell,which methods are useful to ascertain the feasability of such treatment.Monitoring such a therapy, such as, e.g., a therapy that altersassociation of a NAC with a NAC-associated protein in a cell using aneffective agent, can allow for modifications in the therapy to be made,including decreasing the amount of effective agent used in therapy,increasing the amount of effective agent, or using a different effectiveagent. In general, a test sample can be obtained from a subject having apathology characterized by increased or decreased apoptosis, whichsample can be compared to a control sample from a normal subject todetermine whether a cell in the test sample has, for example, increasedor decreased expression of NAC. Preferably, this control sample is aprevious sample from the same patient, thereby providing a directcomparison of changes to the pathology as a result of the therapy. Thelevel of a NAC in a cell can be determined by contacting a sample with areagent such as an anti-NAC antibody or a NAC-associated protein, eitherof which can specifically bind a NAC. For example, the level of a NAC ina cell can determined by well known immunoassay or immunohistochemicalmethods using an anti-NAC antibody (see, for example, Reed et al.,supra, 1992; see, also, Harlow and Lane, supra, (1988)).

[0176] In accordance with another embodiment of the invention, there areprovided methods for determining a prognosis of disease free or overallsurvival in a patient suffering from cancer. For example, it iscontemplated herein that abnormal levels of NAC proteins (either higheror lower) in primary tumor tissue show a high correlation with eitherincreased or decreased tumor recurrence or spread, and thereforeindicates the likelihood of disease free or overall survival. Thus, thepresent invention advantageously provides a significant advancement incancer management because early identification of patients at risk fortumor recurrence or spread will permit aggressive early treatment withsignificantly enhanced potential for survival. Also provided are methodsfor predicting the risk of tumor recurrence or spread in an individualhaving a cancer tumor; methods for screening a cancer patient todetermine the risk of tumor metastasis; and methods for determining theproper course of treatment for a patient suffering from cancer. Thesemethods are carried out by collecting a sample from a patient andcomparing the level of NAC expression in the patient to the level ofexpression in a control or to a reference level of NAC expression asdefined by patient population sampling, tissue culture analysis, or anyother method known for determining reference levels for determination ofdisease prognosis. The level of NAC expression in the patient is thenclassified as higher than the reference level or lower than thereference level, wherein the prognosis of survival or tumor recurrenceis different for patients with higher levels than the prognosis forpatients with lower levels.

[0177] All U.S. patents and all publications mentioned herein areincorporated in their entirety by reference thereto. The invention willnow be described in greater detail by reference to the followingnon-limiting examples.

EXAMPLES

[0178] 1.0 cDNA Cloning.

[0179] Jurkat total RNA was reverse-transcribed to complementary DNAsusing MMLV reverse transcriptase (Stratagene) and random hexanucleotideprimers. Three overlapping cDNA fragments of NAC were amplified from theJurkat complementary DNAs with Turbo Pfu DNA polymerase (Stratagene)using the following oligonucleotide primer sets: primer set 1;5′-CCGAATTCACCATGGCTGGCGGAGCCTGGGGC-3′ (forward; SEQ ID NO:13) and5′-CCGCTCGAGTCAACAGAGGGTTGTGGTGGTCTTG-3′ (reverse; SEQ ID NO:14), primerset 2; 5′-CCCGAATTCGAACCTCGCATAGTCATACTGC-3′ (forward; SEQ ID NO:15) and5′-GTCCCACAACAGAATTCAATCTCAACGGTC-3′ (reverse; SEQ ID NO:16), and primerset 3; 5′-TGTGATGAGAGAAGCGGTGAC-3′ (forward; SEQ ID NO:17) and5′-CCGCTCGAGCAAAGAAGGGTCAGCCAAAGC-3′ (reverse; SEQ ID NO:18). Theresultant cDNA fragments were ligated into mammalian expression vectorpcDNA-myc (Invitrogen, modified as described in Roy et al., EMBO J.16:6914-6925 (1997)) and assembled to full-length cDNA by ligatingfragments 2 and 3 at the EcoRI site to make fragment 4, and by ligatingfragments 1 and 4 at the Bst X1 site, as depicted in FIG. 1A. Sequencinganalysis of the assembled full-length cDNA was carried out, and spliceisoforms (shown as dotted and hatched regions in FIG. 1B) of NAC cloneswere identified. The full-length NAC nucleotide and protein sequences,including two alternatively spliced regions underlined (nucleotides2870-2959 and 3784-3915 of SEQ ID NO:1, respectively), are presented inFIG. 1C. The full length nucleotide sequence of three of the isoforms isset forth in SEQ ID NOs:1, 3 and 5, corresponding to NACβ, NACγ andNACδ, respectively.

[0180] Comparison of NAC to known protein sequences using Clustalmultiple sequence alignment (Thompson et al., Nucleic Acids Research22:4673-4680 (1994)) revealed that the CARD domain of NAC (see, e.g.,residues 1373 to 1473 of SEQ ID NO:2) is similar to numerous CARD domainproteins. Further sequence analysis predicted an α₈β₈ (TIM)-Barrel-likedomain similar to those observed in aldolase and RuBisCo in NAC, locatedon the immediate amino terminal side of the predicted CARD domain (see,e.g., residues 1079 to 1364 of SEQ ID NO:2). Additionally, a portion ofNAC was found to have sequence portions homologous to NB-ARC domains(see, e.g., residues 329 to 547 of SEQ ID NO:2) and a leucine-richrepeat region (see, e.g., residues 808 to 947 of SEQ ID NO:2). Based onits homology to the above proteins the protein of the invention has beentermed a NAC protein, as it is a NB-ARC and CARD domain containingprotein. ClustalW multiple sequence alignment with other NB-ARC and CARDdomain containing proteins confirmed the homology of NAC to otherproteins in both the NB-ARC region (particularly in the P-loop, orWalker A, and Walker B portions) and CARD region (FIG. 1D and FIG. 1E,respectively). This sequence analysis represents the first time a domainresembling a TIM-barrel domain has been identified in a protein thatalso contains a CARD domain, and also the first time a domain resemblinga TIM-barrel domain has been identified in a protein that also containsan NB-ARC domain.

[0181] 2.0 Plasmid Constructions.

[0182] Complementary DNA encoding the CARD domain of NAC was amplifiedfrom Jurkat cDNAs with Turbo Pfu DNA polymerase (Stratagene) and primerset 3 as described above. The resultant PCR fragments were digested withEcoRI and Xho I restriction enzymes and ligated into pGEX-4T1(Pharmacia) and pcDNA-myc vectors. This region of NAC contains twoalternatively spliced isoforms, termed CARD_(L) (amino acid residues1128-1473 of SEQ ID NO:2) and CARD_(S) (amino acid residues 1128-1261and 1306-1473 of SEQ ID NO:2). The region of cDNA encoding NB-ARC domainwas PCR-amplified using primers SEQ ID NO:15 (forward) and SEQ ID NO:14(reverse). The resultant PCR fragment was digested with EcoRI and Xho Irestriction enzymes (yielding a fragment encoding amino acid residues326-551 of SEQ ID NO:2) and ligated into a pGEX-4T1 and pcDNA-mycvectors.

[0183] 3.0 In vitro Protein Binding Assays.

[0184] NB-ARC, CARD_(L), and CARD_(S) in pGEX-4T1 were expressed in XL-1blue E. coli cells (Stratagene), and affinity-purified using glutathione(GSH)-sepharose according to known methods, such as those in CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley and Sons(1999). For GST pull-down assays, purified CARD_(L) and CARD_(S) GSTfusion proteins and GST alone (0.1-0.5 μg immobilized on 10-15 μlGSH-sepharose beads) were incubated with 1 mg/ml of BSA in 100 μl Co-IPbuffer [142.4 mM KCl, 5 mM MgCl₂, 10 mM HEPES (pH 7.4), 0.5 mM EGTA,0.2% NP-40, 1 mM DTT, and 1 mM PMSF] for 30 min. at room temperature.The beads were then incubated with 1 μl of rat reticulocyte lysates(TnT-lysate; Promega, Inc.) containing ³⁵S-labeled, in vitro translatedCARD_(L), CARD_(S), or control protein Skp-1 in 100 μl Co-IP buffersupplemented with 0.5 mg/ml BSA for overnight at 4° C. The beads werewashed four times in 500 μl Co-IP buffer, followed by boiling in 20 μlLaemmli-SDS sample buffer. The eluted proteins were analyzed bySDS-PAGE. The bands of SDS-PAGE gels were detected by fluorography.

[0185] The resultant homodimerization pattern reveals thatCARD_(L)-CARD_(L), CARD_(S)-CARD_(S), and both CARD_(L)-CARD_(S)containing lanes have very strong signals, whereas lanes containingcontrol GST alone and control Skp-1 have negligible signals (FIG. 2A).Thus, CARD domains of the invention NAC show a very strong ability toself-associate in vitro.

[0186] In vitro translated Apaf-1 (lacking its WD domain), CED4, andcontrol Skp-1 proteins were subjected to GST pull-down assay usingGSH-sepharose beads conjugated with GST, GST-CARD_(L), and GST-CARD_(S)as described above. Both lanes containing GST-CARD_(S) and lanescontaining GST-CARD_(L) yielded very strong signals when incubated witheither Apaf-1 (-WD) or CED4, whereas, the controls GST alone and Skp-1again yielded negligible signals (FIG. 2B). Thus, in addition toself-association, CARD domains of the invention NAC demonstrate theability to in vitro associate with other CARD-containing proteins.

[0187] 4.0 Protein Interaction Studies in Yeast.

[0188] EGY48 yeast cells (Saccharomyces cerevisiae: MATα, trpl, ura3,his, leu2::plexApo6-leu2) were transformed with pGilda-CARD_(L) plasmids(his marker) encoding the LexA DNA binding domain fused to: CARD domainsof NAC (CARD_(L)) and caspase-9; pro-caspase-8; Apaf-1 without its WDdomain; Bcl-XL, Bax and Bcl-2 without transmembrane domains. EGY48 werealso transformed with vector pJG4-5 (trpl marker) encoding the abovelisted group of proteins and additionally vRas and FADD as targetproteins, fused to B42 transactivation domain, and the cells weretransformed with a LexA-LacZ reporter plasmid pSH1840 (ura3 marker,), aspreviously described (Durfee et al., 1993; Sato et al., 1995). Sourcesfor cells and plasmids were described previously in U.S. Pat. No.5,632,994, and in Zervous et al., Cell 72:223-232 (1993); Gyuris et al.,Cell 75:791-803 (1993); Golemis et al., In Current Protocols inMolecular Biology (ed. Ausubel et al.; Green Publ.; NY 1994), each ofwhich is incorporated herein by reference. Transformants werereplica-plated on Burkholder's minimal medium (BMM) plates supplementedwith leucine and 2% glucose as previously described (Sato et al., Gene140:291-292 (1994)). Protein-protein interactions were scored by growthof transformants on leucine deficient BMM plates containing 2% galactoseand 1% raffinose.

[0189] Protein-protein interactions were also evaluated usingβ-galactosidase activity assays. Colonies grown on BMM/Leu/Glucoseplates were filter-lifted onto nitrocellulose membranes, and incubatedover-night on BMM/Leu/galactose plates. Yeast cells were lysed bysoaking filters in liquid nitrogen and thawing at room temperature.β-galactosidase activity was measured by incubating the filter in 3.2 mlZ buffer (60 mM, Na₂HPO₄, 40 mM Na₂HPO₄, 10 mM KCl, 1 mM MgSO₄)supplemented with 50 μl X-gal solution (20 mg/ml). Levels ofβ-galactosidase activity were scaled according to the intensity of bluecolor generated for each transformant.

[0190] The results of this experiment showed colonies on leucinedeficient plates for yeast containing NAC-CARD_(L)/LexA fusions togetherwith caspase-9/B42, Apaf-1/B42, and Bax/B42 fusions (FIG. 3). Inaddition, the NAC-CARD_(L)/LexA:caspase-9/B42 andNAC-CARD_(L)/LexA:Apaf-1/B42 cells had significant amounts of LacZactivity. The cells containing the complementary fusionscaspase-9/LexA:NAC-CARD_(L)/B42 and Apaf-1/LexA:NAC-CARD_(L)/B42 alsogrew on leucine deficient plates and showed significant LacZ activity.Thus all four indicators of protein:protein interaction confirmed thatthe CARD_(L) domain of NAC interacts with the CARD domains of caspase-9and with Apaf-1. Partial indication of the protein:protein interactionswith NAC-CARD_(L) were observed for Bax, caspase-8, Bcl-XL and Bcl-2,suggesting that a broad range of CARD domain proteins also interact withthe CARD domain of NAC.

[0191] Similar two-hybrid interaction experiments have been performedusing the CARD domain of the CARD-X protein. Table I summarizes theresults of the two-hybrid experiments wherein a fusion proteincontaining the DNA-binding domain of the LexA protein expressed from thepGilda plasmid and a CARD domain from CARD-X or several otherCARD-containing proteins, including CARDIAK, NAC (CARD_(L)), Apaf-1,caspases-2, 9, and 11, were expressed in the sames cells as CARD domainsfrom CARD-X, CARDIAK, NAC(CARD_(L)), caspase-9 and cIAP-2, expressed asfusion proteins with a transactivation domain from the B42 protein fromthe pJG4-5 plasmid, as described above. As shown, the CARD domain ofCARD-X interacted with itself but not with the CARD domains of otherproteins. TABLE I Yeast Two Hybrid Analysis of CARD-X:CARD interactionspGilda pJG4-5 Results 1 CARD-X CARD CARD-X-CARD +++ 2 CARD-X CARDCARDIAK − 3 CARD-X CARD NAC-CARD_(L) − 4 CARD-X CARD Caspase-9 CARD − 5CARD-X CARD cIAP-2 − 6 CARDIAK CARD-X CARD − 7 NAC-CARD_(L) CARD-X CARD− 8 APAF C3 + C4 CARD-X CARD − 9 Caspase-2 CARD-X CARD − 10 Caspase-11CARD-X CARD − 11 Caspase 9-C-terminus CARD-X CARD − 12 CARDIAK CARDIAK++++

[0192] 5.0 Self-Association of NB-ARC domain of NAC.

[0193] In vitro translated, ³⁵S-labeled rat reticulocyte lysates (1 μl)containing NB-ARC or Skp-1 (used as a control) were incubated withGSH-sepharose beads conjugated with purified GST-NB-ARC or GST alone forGST pull-down assay, resolved on SDS-PAGE and visualized by fluorographyas described above. One tenth of input were loaded for NB-ARC or Skp-1as controls. In this assay, the NB-ARC-containing fragment of NACdemonstrates a strong ability to homodimerize (FIG. 4).

[0194] The ability to self-associate and to bind other known CARDdomains establishes the CARD domains of NAC, CARD_(S) and CARD_(L), ascapable of the same protein-protein interactions observed in other knownCARD domains. The ability of CARD-X to self-associate also establishesthis protein as having the same protein-protein interaction propertiesof known CARD proteins. Thus two isoforms of a new human CARD domainhave been characterized, and a highly related sequence of another humanprotein CARD-X has also been characterized. In addition, the ability ofthe putative NB-ARC domain of NAC has been shown to both self-associate,establishing this domain as capable of the same protein-proteininteractions observed in other known NB-ARC domains. Therefore, the NACprotein has been demonstrated to contain both a functional CARD domainand a functional NB-ARC domain.

[0195] 6.0 Protein-Protein Interactions of NAC.

[0196] Transient transfection of 293T, a human embryonic kidneyfibroblast cell line, were conducted using SuperFect reagents (Qiagen)according to manufacturer's instructions. The cDNA fragments encodingfull-length CED4 and the truncated form of Apaf-1 (Apaf-1ΔWD) comprisingamino acids 1-420 of the human Apaf-1 protein were amplified by PCR andsubcloned into pcDNA3HA at EcoRI and Xho I sites. Expression plasmidsencoding catalytically inactive forms of pro-Casp8 [pro-Casp8 (C/A)] wasprepared by replacing Cys 377 with an Ala using site-directedmutagenesis and pro-Casp9 [pro-Casp9 (C/A)] has been describedpreviously, Cardone et al., Science 282:1318-1321 (1998)). 293T cellswere transiently transfected with an expression plasmid (2 μg) encodingHA-tagged human Apaf-1ΔWD, CED4, pro-Casp8 (C/A) or C-TerminalFlag-tagged pro-Casp9 (C/A) in the presence or absence of a plasmid (2μg) encoding myc-tagged NAC (encoding amino acid residues 1-1261 and1306-1473 of SEQ ID NO:2). After 24 hr growth in culture, transfectedcells were collected and lysed in Co-IP buffer [142.4 mM KCl, 5 mMMgCl₂, 10 mM HEPES (pH 7.4), 0.5 mM EGTA, 0.1% NP-40, and 1 mM DTT]supplemented with 12.5 mM β-glycerolphosphate, 2 mM NaF, 1 mM Na₃VO₄, 1mM PMSF, and 1× protenase inhibitor mix (Boehringer Mannheim). Celllysates were clarified by microcentrifugation and subjected toimmunoprecipitation using either a mouse monoclonal antibody to myc(Santa Cruz Biotechnologies, Inc) or a control mouse IgG. Proteins fromthe immune complexes were resolved by SDS-PAGE, transferred tonitrocellulose membranes, and subjected to immunoblot analysis usinganti-HA antibodies followed by anti-myc antibodies using a standardWestern blotting procedure and ECL reagents from Amersham-PharmaciaBiotechnologies, Inc. (Krajewski et al., Proc. Natl. Acad. Sci. USA96:5752-5757 (1999)).

[0197] The results show that NAC of the invention interacts with otherNB-ARC and CARD-containing proteins, Apaf-1 (FIG. 5A) and CED-4 (FIG.5B), and additionally with caspase-8 (FIG. 6A), but not with caspase-9(FIG. 6B). This is in contrast with the observed interaction betweencaspase-9 and the CARD_(L) domain of NAC from the above described yeasttwo-hybrid assay. This may be due to the regulation of the full-lengthNAC in terms of its ability to interact with pro-caspase-9 such that NACis in either a latent (off) or active (on) conformation, analogous toApaf-1 which binds pro-caspase-9 only when cytochrome c is produced toinduce a conformational change in Apaf-1. As with NAC, if only the CARDdomain of Apaf-1 is expressed, it will bind to pro-caspase-9independently of the coactivator, cytochrome c (Qin et al., Nature399:549-557 (1999)).

[0198] Although the invention has been described with reference to theexamples above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 18 1 4422 DNA Homo sapiens CDS (1)..(4422) 1 atg gct ggc gga gcc tggggc cgc ctg gcc tgt tac ttg gag ttc ctg 48 Met Ala Gly Gly Ala Trp GlyArg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15 aag aag gag gag ctg aaggag ttc cag ctt ctg ctc gcc aat aaa gcg 96 Lys Lys Glu Glu Leu Lys GluPhe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 cac tcc agg agc tct tcg ggtgag aca ccc gct cag cca gag aag acg 144 His Ser Arg Ser Ser Ser Gly GluThr Pro Ala Gln Pro Glu Lys Thr 35 40 45 agt ggc atg gag gtg gcc tcg tacctg gtg gct cag tat ggg gag cag 192 Ser Gly Met Glu Val Ala Ser Tyr LeuVal Ala Gln Tyr Gly Glu Gln 50 55 60 cgg gcc tgg gac cta gcc ctc cat acctgg gag cag atg ggg ctg agg 240 Arg Ala Trp Asp Leu Ala Leu His Thr TrpGlu Gln Met Gly Leu Arg 65 70 75 80 tca ctg tgc gcc caa gcc cag gaa ggggca ggc cac tct ccc tca ttc 288 Ser Leu Cys Ala Gln Ala Gln Glu Gly AlaGly His Ser Pro Ser Phe 85 90 95 ccc tac agc cca agt gaa ccc cac ctg gggtct ccc agc caa ccc acc 336 Pro Tyr Ser Pro Ser Glu Pro His Leu Gly SerPro Ser Gln Pro Thr 100 105 110 tcc acc gca gtg cta atg ccc tgg atc catgaa ttg ccg gcg ggg tgc 384 Ser Thr Ala Val Leu Met Pro Trp Ile His GluLeu Pro Ala Gly Cys 115 120 125 acc cag ggc tca gag aga agg gtt ttg agacag ctg cct gac aca tct 432 Thr Gln Gly Ser Glu Arg Arg Val Leu Arg GlnLeu Pro Asp Thr Ser 130 135 140 gga cgc cgc tgg aga gaa atc tct gcc tcactc ctc tac caa gct ctt 480 Gly Arg Arg Trp Arg Glu Ile Ser Ala Ser LeuLeu Tyr Gln Ala Leu 145 150 155 160 cca agc tcc cca gac cat gag tct ccaagc cag gag tca ccc aac gcc 528 Pro Ser Ser Pro Asp His Glu Ser Pro SerGln Glu Ser Pro Asn Ala 165 170 175 ccc aca tcc aca gca gtg ctg ggg agctgg gga tcc cca cct cag ccc 576 Pro Thr Ser Thr Ala Val Leu Gly Ser TrpGly Ser Pro Pro Gln Pro 180 185 190 agc cta gca ccc aga gag cag gag gctcct ggg acc caa tgg cct ctg 624 Ser Leu Ala Pro Arg Glu Gln Glu Ala ProGly Thr Gln Trp Pro Leu 195 200 205 gat gaa acg tca gga att tac tac acagaa atc aga gaa aga gag aga 672 Asp Glu Thr Ser Gly Ile Tyr Tyr Thr GluIle Arg Glu Arg Glu Arg 210 215 220 gag aaa tca gag aaa ggc agg ccc ccatgg gca gcg gtg gta gga acg 720 Glu Lys Ser Glu Lys Gly Arg Pro Pro TrpAla Ala Val Val Gly Thr 225 230 235 240 ccc cca cag gcg cac acc agc ctacag ccc cac cac cac cca tgg gag 768 Pro Pro Gln Ala His Thr Ser Leu GlnPro His His His Pro Trp Glu 245 250 255 cct tct gtg aga gag agc ctc tgttcc aca tgg ccc tgg aaa aat gag 816 Pro Ser Val Arg Glu Ser Leu Cys SerThr Trp Pro Trp Lys Asn Glu 260 265 270 gat ttt aac caa aaa ttc aca cagctg cta ctt cta caa aga cct cac 864 Asp Phe Asn Gln Lys Phe Thr Gln LeuLeu Leu Leu Gln Arg Pro His 275 280 285 ccc aga agc caa gat ccc ctg gtcaag aga agc tgg cct gat tat gtg 912 Pro Arg Ser Gln Asp Pro Leu Val LysArg Ser Trp Pro Asp Tyr Val 290 295 300 gag gag aat cga gga cat tta attgag atc aga gac tta ttt ggc cca 960 Glu Glu Asn Arg Gly His Leu Ile GluIle Arg Asp Leu Phe Gly Pro 305 310 315 320 ggc ctg gat acc caa gaa cctcgc ata gtc ata ctg cag ggg gct gct 1008 Gly Leu Asp Thr Gln Glu Pro ArgIle Val Ile Leu Gln Gly Ala Ala 325 330 335 gga att ggg aag tca aca ctggcc agg cag gtg aag gaa gcc tgg ggg 1056 Gly Ile Gly Lys Ser Thr Leu AlaArg Gln Val Lys Glu Ala Trp Gly 340 345 350 aga ggc cag ctg tat ggg gaccgc ttc cag cat gtc ttc tac ttc agc 1104 Arg Gly Gln Leu Tyr Gly Asp ArgPhe Gln His Val Phe Tyr Phe Ser 355 360 365 tgc aga gag ctg gcc cag tccaag gtg gtg agt ctc gct gag ctc atc 1152 Cys Arg Glu Leu Ala Gln Ser LysVal Val Ser Leu Ala Glu Leu Ile 370 375 380 gga aaa gat ggg aca gcc actccg gct ccc att aga cag atc ctg tct 1200 Gly Lys Asp Gly Thr Ala Thr ProAla Pro Ile Arg Gln Ile Leu Ser 385 390 395 400 agg cca gag cgg ctg ctcttc atc ctc gat ggt gta gat gag cca gga 1248 Arg Pro Glu Arg Leu Leu PheIle Leu Asp Gly Val Asp Glu Pro Gly 405 410 415 tgg gtc ttg cag gag ccgagt tct gag ctc tgt ctg cac tgg agc cag 1296 Trp Val Leu Gln Glu Pro SerSer Glu Leu Cys Leu His Trp Ser Gln 420 425 430 cca cag ccg gcg gat gcactg ctg ggc agt ttg ctg ggg aaa act ata 1344 Pro Gln Pro Ala Asp Ala LeuLeu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445 ctt ccc gag gca tcc ttcctg atc acg gct cgg acc aca gct ctg cag 1392 Leu Pro Glu Ala Ser Phe LeuIle Thr Ala Arg Thr Thr Ala Leu Gln 450 455 460 aac ctc att cct tct ttggag cag gca cgt tgg gta gag gtc ctg ggg 1440 Asn Leu Ile Pro Ser Leu GluGln Ala Arg Trp Val Glu Val Leu Gly 465 470 475 480 ttc tct gag tcc agcagg aag gaa tat ttc tac aga tat ttc aca gat 1488 Phe Ser Glu Ser Ser ArgLys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485 490 495 gaa agg caa gca attaga gcc ttt agg ttg gtc aaa tca aac aaa gag 1536 Glu Arg Gln Ala Ile ArgAla Phe Arg Leu Val Lys Ser Asn Lys Glu 500 505 510 ctc tgg gcc ctg tgtctt gtg ccc tgg gtg tcc tgg ctg gcc tgc act 1584 Leu Trp Ala Leu Cys LeuVal Pro Trp Val Ser Trp Leu Ala Cys Thr 515 520 525 tgc ctg atg cag cagatg aag cgg aag gaa aaa ctc aca ctg act tcc 1632 Cys Leu Met Gln Gln MetLys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535 540 aag acc acc aca accctc tgt cta cat tac ctt gcc cag gct ctc caa 1680 Lys Thr Thr Thr Thr LeuCys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560 gct cag cca ttggga ccc cag ctc aga gac ctc tgc tct ctg gct gct 1728 Ala Gln Pro Leu GlyPro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570 575 gag ggc atc tggcaa aaa aag acc ctt ttc agt cca gat gac ctc agg 1776 Glu Gly Ile Trp GlnLys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580 585 590 aag cat ggg ttagat ggg gcc atc atc tcc acc ttc ttg aag atg ggt 1824 Lys His Gly Leu AspGly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595 600 605 att ctt caa gagcac ccc atc cct ctg agc tac agc ttc att cac ctc 1872 Ile Leu Gln Glu HisPro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu 610 615 620 tgt ttc caa gagttc ttt gca gca atg tcc tat gtc ttg gag gat gag 1920 Cys Phe Gln Glu PhePhe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625 630 635 640 aag ggg agaggt aaa cat tct aat tgc atc ata gat ttg gaa aag acg 1968 Lys Gly Arg GlyLys His Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645 650 655 cta gaa gcatat gga ata cat ggc ctg ttt ggg gca tca acc aca cgt 2016 Leu Glu Ala TyrGly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg 660 665 670 ttc cta ttgggc ctg tta agt gat gag ggg gag aga gag atg gag aac 2064 Phe Leu Leu GlyLeu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn 675 680 685 atc ttt cactgc cgg ctg tct cag ggg agg aac ctg atg cag tgg gtc 2112 Ile Phe His CysArg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val 690 695 700 ccg tcc ctgcag ctg ctg ctg cag cca cac tct ctg gag tcc ctc cac 2160 Pro Ser Leu GlnLeu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His 705 710 715 720 tgc ttgtac gag act cgg aac aaa acg ttc ctg aca caa gtg atg gcc 2208 Cys Leu TyrGlu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val Met Ala 725 730 735 cat ttcgaa gaa atg ggc atg tgt gta gaa aca gac atg gag ctc tta 2256 His Phe GluGlu Met Gly Met Cys Val Glu Thr Asp Met Glu Leu Leu 740 745 750 gtg tgcact ttc tgc att aaa ttc agc cgc cac gtg aag aag ctt cag 2304 Val Cys ThrPhe Cys Ile Lys Phe Ser Arg His Val Lys Lys Leu Gln 755 760 765 ctg attgag ggc agg cag cac aga tca aca tgg agc ccc acc atg gta 2352 Leu Ile GluGly Arg Gln His Arg Ser Thr Trp Ser Pro Thr Met Val 770 775 780 gtc ctgttc agg tgg gtc cca gtc aca gat gcc tat tgg cag att ctc 2400 Val Leu PheArg Trp Val Pro Val Thr Asp Ala Tyr Trp Gln Ile Leu 785 790 795 800 ttctcc gtc ctc aag gtc acc aga aac ctg aag gag ctg gac cta agt 2448 Phe SerVal Leu Lys Val Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805 810 815 ggaaac tcg ctg agc cac tct gca gtg aag agt ctt tgt aag acc ctg 2496 Gly AsnSer Leu Ser His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820 825 830 agacgc cct cgc tgc ctc ctg gag acc ctg cgg ttg gct ggc tgt ggc 2544 Arg ArgPro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly 835 840 845 ctcaca gct gag gac tgc aag gac ctt gcc ttt ggg ctg aga gcc aac 2592 Leu ThrAla Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg Ala Asn 850 855 860 cagacc ctg acc gag ctg gac ctg agc ttc aat gtg ctc acg gat gct 2640 Gln ThrLeu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu Thr Asp Ala 865 870 875 880gga gcc aaa cac ctt tgc cag aga ctg aga cag ccg agc tgc aag cta 2688 GlyAla Lys His Leu Cys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu 885 890 895cag cga ctg cag ctg gtc agc tgt ggc ctc acg tct gac tgc tgc cag 2736 GlnArg Leu Gln Leu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln 900 905 910gac ctg gcc tct gtg ctt agt gcc agc ccc agc ctg aag gag cta gac 2784 AspLeu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu Lys Glu Leu Asp 915 920 925ctg cag cag aac aac ctg gat gac gtt ggc gtg cga ctg ctc tgt gag 2832 LeuGln Gln Asn Asn Leu Asp Asp Val Gly Val Arg Leu Leu Cys Glu 930 935 940ggg ctc agg cat cct gcc tgc aaa ctc ata cgc ctg ggg ctg gac cag 2880 GlyLeu Arg His Pro Ala Cys Lys Leu Ile Arg Leu Gly Leu Asp Gln 945 950 955960 aca act ctg agt gat gag atg agg cag gaa ctg agg gcc ctg gag cag 2928Thr Thr Leu Ser Asp Glu Met Arg Gln Glu Leu Arg Ala Leu Glu Gln 965 970975 gag aaa cct cag ctg ctc atc ttc agc aga cgg aaa cca agt gtg atg 2976Glu Lys Pro Gln Leu Leu Ile Phe Ser Arg Arg Lys Pro Ser Val Met 980 985990 acc cct act gag ggc ctg gat acg gga gag atg agt aat agc aca tcc 3024Thr Pro Thr Glu Gly Leu Asp Thr Gly Glu Met Ser Asn Ser Thr Ser 995 10001005 tca ctc aag cgg cag aga ctc gga tca gag agg gcg gct tcc cat gtt3072 Ser Leu Lys Arg Gln Arg Leu Gly Ser Glu Arg Ala Ala Ser His Val1010 1015 1020 gct cag gct aat ctc aaa ctc ctg gac gtg agc aag atc ttccca att 3120 Ala Gln Ala Asn Leu Lys Leu Leu Asp Val Ser Lys Ile Phe ProIle 1025 1030 1035 1040 gct gag att gca gag gaa agc tcc cca gag gta gtaccg gtg gaa ctc 3168 Ala Glu Ile Ala Glu Glu Ser Ser Pro Glu Val Val ProVal Glu Leu 1045 1050 1055 ttg tgc gtg cct tct cct gcc tct caa ggg gacctg cat acg aag cct 3216 Leu Cys Val Pro Ser Pro Ala Ser Gln Gly Asp LeuHis Thr Lys Pro 1060 1065 1070 ttg ggg act gac gat gac ttc tgg ggc cccacg ggg cct gtg gct act 3264 Leu Gly Thr Asp Asp Asp Phe Trp Gly Pro ThrGly Pro Val Ala Thr 1075 1080 1085 gag gta gtt gac aaa gaa aag aac ttgtac cga gtt cac ttc cct gta 3312 Glu Val Val Asp Lys Glu Lys Asn Leu TyrArg Val His Phe Pro Val 1090 1095 1100 gct ggc tcc tac cgc tgg ccc aacacg ggt ctc tgc ttt gtg atg aga 3360 Ala Gly Ser Tyr Arg Trp Pro Asn ThrGly Leu Cys Phe Val Met Arg 1105 1110 1115 1120 gaa gcg gtg acc gtt gagatt gaa ttc tgt gtg tgg gac cag ttc ctg 3408 Glu Ala Val Thr Val Glu IleGlu Phe Cys Val Trp Asp Gln Phe Leu 1125 1130 1135 ggt gag atc aac ccacag cac agc tgg atg gtg gca ggg cct ctg ctg 3456 Gly Glu Ile Asn Pro GlnHis Ser Trp Met Val Ala Gly Pro Leu Leu 1140 1145 1150 gac atc aag gctgag cct gga gct gtg gaa gct gtg cac ctc cct cac 3504 Asp Ile Lys Ala GluPro Gly Ala Val Glu Ala Val His Leu Pro His 1155 1160 1165 ttt gtg gctctc caa ggg ggc cat gtg gac aca tcc ctg ttc caa atg 3552 Phe Val Ala LeuGln Gly Gly His Val Asp Thr Ser Leu Phe Gln Met 1170 1175 1180 gcc cacttt aaa gag gag ggg atg ctc ctg gag aag cca gcc agg gtg 3600 Ala His PheLys Glu Glu Gly Met Leu Leu Glu Lys Pro Ala Arg Val 1185 1190 1195 1200gag ctg cat cac ata gtt ctg gaa aac ccc agc ttc tcc ccc ttg gga 3648 GluLeu His His Ile Val Leu Glu Asn Pro Ser Phe Ser Pro Leu Gly 1205 12101215 gtc ctc ctg aaa atg atc cat aat gcc ctg cgc ttc att ccc gtc acc3696 Val Leu Leu Lys Met Ile His Asn Ala Leu Arg Phe Ile Pro Val Thr1220 1225 1230 tct gtg gtg ttg ctt tac cac cgc gtc cat cct gag gaa gtcacc ttc 3744 Ser Val Val Leu Leu Tyr His Arg Val His Pro Glu Glu Val ThrPhe 1235 1240 1245 cac ctc tac ctg atc cca agt gac tgc tcc att cgg aaggcc ata gat 3792 His Leu Tyr Leu Ile Pro Ser Asp Cys Ser Ile Arg Lys AlaIle Asp 1250 1255 1260 gat cta gaa atg aaa ttc cag ttt gtg cga atc cacaag cca ccc ccg 3840 Asp Leu Glu Met Lys Phe Gln Phe Val Arg Ile His LysPro Pro Pro 1265 1270 1275 1280 ctg acc cca ctt tat atg ggc tgt cgt tacact gtg tct ggg tct ggt 3888 Leu Thr Pro Leu Tyr Met Gly Cys Arg Tyr ThrVal Ser Gly Ser Gly 1285 1290 1295 tca ggg atg ctg gaa ata ctc ccc aaggaa ctg gag ctc tgc tat cga 3936 Ser Gly Met Leu Glu Ile Leu Pro Lys GluLeu Glu Leu Cys Tyr Arg 1300 1305 1310 agc cct gga gaa gac cag ctg ttctcg gag ttc tac gtt ggc cac ttg 3984 Ser Pro Gly Glu Asp Gln Leu Phe SerGlu Phe Tyr Val Gly His Leu 1315 1320 1325 gga tca ggg atc agg ctg caagtg aaa gac aag aaa gat gag act ctg 4032 Gly Ser Gly Ile Arg Leu Gln ValLys Asp Lys Lys Asp Glu Thr Leu 1330 1335 1340 gtg tgg gag gcc ttg gtgaaa cca gga gat ctc atg cct gca act act 4080 Val Trp Glu Ala Leu Val LysPro Gly Asp Leu Met Pro Ala Thr Thr 1345 1350 1355 1360 ctg atc cct ccagcc cgc ata gcc gta cct tca cct ctg gat gcc ccg 4128 Leu Ile Pro Pro AlaArg Ile Ala Val Pro Ser Pro Leu Asp Ala Pro 1365 1370 1375 cag ttg ctgcac ttt gtg gac cag tat cga gag cag ctg ata gcc cga 4176 Gln Leu Leu HisPhe Val Asp Gln Tyr Arg Glu Gln Leu Ile Ala Arg 1380 1385 1390 gtg acatcg gtg gag gtt gtc ttg gac aaa ctg cat gga cag gtg ctg 4224 Val Thr SerVal Glu Val Val Leu Asp Lys Leu His Gly Gln Val Leu 1395 1400 1405 agccag gag cag tac gag agg gtg ctg gct gag aac acg agg ccc agc 4272 Ser GlnGlu Gln Tyr Glu Arg Val Leu Ala Glu Asn Thr Arg Pro Ser 1410 1415 1420cag atg cgg aag ctg ttc agc ttg agc cag tcc tgg gac cgg aag tgc 4320 GlnMet Arg Lys Leu Phe Ser Leu Ser Gln Ser Trp Asp Arg Lys Cys 1425 14301435 1440 aaa gat gga ctc tac caa gcc ctg aag gag acc cat cct cac ctcatt 4368 Lys Asp Gly Leu Tyr Gln Ala Leu Lys Glu Thr His Pro His Leu Ile1445 1450 1455 atg gaa ctc tgg gag aag ggc agc aaa aag gga ctc ctg ccactc agc 4416 Met Glu Leu Trp Glu Lys Gly Ser Lys Lys Gly Leu Leu Pro LeuSer 1460 1465 1470 agc tga 4422 Ser 2 1473 PRT Homo sapiens 2 Met AlaGly Gly Ala Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15 LysLys Glu Glu Leu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 HisSer Arg Ser Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45 SerGly Met Glu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 ArgAla Trp Asp Leu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80Ser Leu Cys Ala Gln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 90 95Pro Tyr Ser Pro Ser Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100 105110 Ser Thr Ala Val Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115120 125 Thr Gln Gly Ser Glu Arg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser130 135 140 Gly Arg Arg Trp Arg Glu Ile Ser Ala Ser Leu Leu Tyr Gln AlaLeu 145 150 155 160 Pro Ser Ser Pro Asp His Glu Ser Pro Ser Gln Glu SerPro Asn Ala 165 170 175 Pro Thr Ser Thr Ala Val Leu Gly Ser Trp Gly SerPro Pro Gln Pro 180 185 190 Ser Leu Ala Pro Arg Glu Gln Glu Ala Pro GlyThr Gln Trp Pro Leu 195 200 205 Asp Glu Thr Ser Gly Ile Tyr Tyr Thr GluIle Arg Glu Arg Glu Arg 210 215 220 Glu Lys Ser Glu Lys Gly Arg Pro ProTrp Ala Ala Val Val Gly Thr 225 230 235 240 Pro Pro Gln Ala His Thr SerLeu Gln Pro His His His Pro Trp Glu 245 250 255 Pro Ser Val Arg Glu SerLeu Cys Ser Thr Trp Pro Trp Lys Asn Glu 260 265 270 Asp Phe Asn Gln LysPhe Thr Gln Leu Leu Leu Leu Gln Arg Pro His 275 280 285 Pro Arg Ser GlnAsp Pro Leu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300 Glu Glu AsnArg Gly His Leu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320 GlyLeu Asp Thr Gln Glu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330 335Gly Ile Gly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345350 Arg Gly Gln Leu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355360 365 Cys Arg Glu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu Leu Ile370 375 380 Gly Lys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln Ile LeuSer 385 390 395 400 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val AspGlu Pro Gly 405 410 415 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu Cys LeuHis Trp Ser Gln 420 425 430 Pro Gln Pro Ala Asp Ala Leu Leu Gly Ser LeuLeu Gly Lys Thr Ile 435 440 445 Leu Pro Glu Ala Ser Phe Leu Ile Thr AlaArg Thr Thr Ala Leu Gln 450 455 460 Asn Leu Ile Pro Ser Leu Glu Gln AlaArg Trp Val Glu Val Leu Gly 465 470 475 480 Phe Ser Glu Ser Ser Arg LysGlu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485 490 495 Glu Arg Gln Ala Ile ArgAla Phe Arg Leu Val Lys Ser Asn Lys Glu 500 505 510 Leu Trp Ala Leu CysLeu Val Pro Trp Val Ser Trp Leu Ala Cys Thr 515 520 525 Cys Leu Met GlnGln Met Lys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535 540 Lys Thr ThrThr Thr Leu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560 AlaGln Pro Leu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570 575Glu Gly Ile Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580 585590 Lys His Gly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595600 605 Ile Leu Gln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu610 615 620 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu AspGlu 625 630 635 640 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile Asp LeuGlu Lys Thr 645 650 655 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe Gly AlaSer Thr Thr Arg 660 665 670 Phe Leu Leu Gly Leu Leu Ser Asp Glu Gly GluArg Glu Met Glu Asn 675 680 685 Ile Phe His Cys Arg Leu Ser Gln Gly ArgAsn Leu Met Gln Trp Val 690 695 700 Pro Ser Leu Gln Leu Leu Leu Gln ProHis Ser Leu Glu Ser Leu His 705 710 715 720 Cys Leu Tyr Glu Thr Arg AsnLys Thr Phe Leu Thr Gln Val Met Ala 725 730 735 His Phe Glu Glu Met GlyMet Cys Val Glu Thr Asp Met Glu Leu Leu 740 745 750 Val Cys Thr Phe CysIle Lys Phe Ser Arg His Val Lys Lys Leu Gln 755 760 765 Leu Ile Glu GlyArg Gln His Arg Ser Thr Trp Ser Pro Thr Met Val 770 775 780 Val Leu PheArg Trp Val Pro Val Thr Asp Ala Tyr Trp Gln Ile Leu 785 790 795 800 PheSer Val Leu Lys Val Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805 810 815Gly Asn Ser Leu Ser His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820 825830 Arg Arg Pro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly 835840 845 Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg Ala Asn850 855 860 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu Thr AspAla 865 870 875 880 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln Pro SerCys Lys Leu 885 890 895 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu Thr SerAsp Cys Cys Gln 900 905 910 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro SerLeu Lys Glu Leu Asp 915 920 925 Leu Gln Gln Asn Asn Leu Asp Asp Val GlyVal Arg Leu Leu Cys Glu 930 935 940 Gly Leu Arg His Pro Ala Cys Lys LeuIle Arg Leu Gly Leu Asp Gln 945 950 955 960 Thr Thr Leu Ser Asp Glu MetArg Gln Glu Leu Arg Ala Leu Glu Gln 965 970 975 Glu Lys Pro Gln Leu LeuIle Phe Ser Arg Arg Lys Pro Ser Val Met 980 985 990 Thr Pro Thr Glu GlyLeu Asp Thr Gly Glu Met Ser Asn Ser Thr Ser 995 1000 1005 Ser Leu LysArg Gln Arg Leu Gly Ser Glu Arg Ala Ala Ser His Val 1010 1015 1020 AlaGln Ala Asn Leu Lys Leu Leu Asp Val Ser Lys Ile Phe Pro Ile 1025 10301035 1040 Ala Glu Ile Ala Glu Glu Ser Ser Pro Glu Val Val Pro Val GluLeu 1045 1050 1055 Leu Cys Val Pro Ser Pro Ala Ser Gln Gly Asp Leu HisThr Lys Pro 1060 1065 1070 Leu Gly Thr Asp Asp Asp Phe Trp Gly Pro ThrGly Pro Val Ala Thr 1075 1080 1085 Glu Val Val Asp Lys Glu Lys Asn LeuTyr Arg Val His Phe Pro Val 1090 1095 1100 Ala Gly Ser Tyr Arg Trp ProAsn Thr Gly Leu Cys Phe Val Met Arg 1105 1110 1115 1120 Glu Ala Val ThrVal Glu Ile Glu Phe Cys Val Trp Asp Gln Phe Leu 1125 1130 1135 Gly GluIle Asn Pro Gln His Ser Trp Met Val Ala Gly Pro Leu Leu 1140 1145 1150Asp Ile Lys Ala Glu Pro Gly Ala Val Glu Ala Val His Leu Pro His 11551160 1165 Phe Val Ala Leu Gln Gly Gly His Val Asp Thr Ser Leu Phe GlnMet 1170 1175 1180 Ala His Phe Lys Glu Glu Gly Met Leu Leu Glu Lys ProAla Arg Val 1185 1190 1195 1200 Glu Leu His His Ile Val Leu Glu Asn ProSer Phe Ser Pro Leu Gly 1205 1210 1215 Val Leu Leu Lys Met Ile His AsnAla Leu Arg Phe Ile Pro Val Thr 1220 1225 1230 Ser Val Val Leu Leu TyrHis Arg Val His Pro Glu Glu Val Thr Phe 1235 1240 1245 His Leu Tyr LeuIle Pro Ser Asp Cys Ser Ile Arg Lys Ala Ile Asp 1250 1255 1260 Asp LeuGlu Met Lys Phe Gln Phe Val Arg Ile His Lys Pro Pro Pro 1265 1270 12751280 Leu Thr Pro Leu Tyr Met Gly Cys Arg Tyr Thr Val Ser Gly Ser Gly1285 1290 1295 Ser Gly Met Leu Glu Ile Leu Pro Lys Glu Leu Glu Leu CysTyr Arg 1300 1305 1310 Ser Pro Gly Glu Asp Gln Leu Phe Ser Glu Phe TyrVal Gly His Leu 1315 1320 1325 Gly Ser Gly Ile Arg Leu Gln Val Lys AspLys Lys Asp Glu Thr Leu 1330 1335 1340 Val Trp Glu Ala Leu Val Lys ProGly Asp Leu Met Pro Ala Thr Thr 1345 1350 1355 1360 Leu Ile Pro Pro AlaArg Ile Ala Val Pro Ser Pro Leu Asp Ala Pro 1365 1370 1375 Gln Leu LeuHis Phe Val Asp Gln Tyr Arg Glu Gln Leu Ile Ala Arg 1380 1385 1390 ValThr Ser Val Glu Val Val Leu Asp Lys Leu His Gly Gln Val Leu 1395 14001405 Ser Gln Glu Gln Tyr Glu Arg Val Leu Ala Glu Asn Thr Arg Pro Ser1410 1415 1420 Gln Met Arg Lys Leu Phe Ser Leu Ser Gln Ser Trp Asp ArgLys Cys 1425 1430 1435 1440 Lys Asp Gly Leu Tyr Gln Ala Leu Lys Glu ThrHis Pro His Leu Ile 1445 1450 1455 Met Glu Leu Trp Glu Lys Gly Ser LysLys Gly Leu Leu Pro Leu Ser 1460 1465 1470 Ser 3 4200 DNA Homo sapiensCDS (1)..(4197) 3 atg gct ggc gga gcc tgg ggc cgc ctg gcc tgt tac ttggag ttc ctg 48 Met Ala Gly Gly Ala Trp Gly Arg Leu Ala Cys Tyr Leu GluPhe Leu 1 5 10 15 aag aag gag gag ctg aag gag ttc cag ctt ctg ctc gccaat aaa gcg 96 Lys Lys Glu Glu Leu Lys Glu Phe Gln Leu Leu Leu Ala AsnLys Ala 20 25 30 cac tcc agg agc tct tcg ggt gag aca ccc gct cag cca gagaag acg 144 His Ser Arg Ser Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu LysThr 35 40 45 agt ggc atg gag gtg gcc tcg tac ctg gtg gct cag tat ggg gagcag 192 Ser Gly Met Glu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln50 55 60 cgg gcc tgg gac cta gcc ctc cat acc tgg gag cag atg ggg ctg agg240 Arg Ala Trp Asp Leu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 6570 75 80 tca ctg tgc gcc caa gcc cag gaa ggg gca ggc cac tct ccc tca ttc288 Ser Leu Cys Ala Gln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 8590 95 ccc tac agc cca agt gaa ccc cac ctg ggg tct ccc agc caa ccc acc336 Pro Tyr Ser Pro Ser Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100105 110 tcc acc gca gtg cta atg ccc tgg atc cat gaa ttg ccg gcg ggg tgc384 Ser Thr Ala Val Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115120 125 acc cag ggc tca gag aga agg gtt ttg aga cag ctg cct gac aca tct432 Thr Gln Gly Ser Glu Arg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser 130135 140 gga cgc cgc tgg aga gaa atc tct gcc tca ctc ctc tac caa gct ctt480 Gly Arg Arg Trp Arg Glu Ile Ser Ala Ser Leu Leu Tyr Gln Ala Leu 145150 155 160 cca agc tcc cca gac cat gag tct cca agc cag gag tca ccc aacgcc 528 Pro Ser Ser Pro Asp His Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala165 170 175 ccc aca tcc aca gca gtg ctg ggg agc tgg gga tcc cca cct cagccc 576 Pro Thr Ser Thr Ala Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro180 185 190 agc cta gca ccc aga gag cag gag gct cct ggg acc caa tgg cctctg 624 Ser Leu Ala Pro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu195 200 205 gat gaa acg tca gga att tac tac aca gaa atc aga gaa aga gagaga 672 Asp Glu Thr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg210 215 220 gag aaa tca gag aaa ggc agg ccc cca tgg gca gcg gtg gta ggaacg 720 Glu Lys Ser Glu Lys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr225 230 235 240 ccc cca cag gcg cac acc agc cta cag ccc cac cac cac ccatgg gag 768 Pro Pro Gln Ala His Thr Ser Leu Gln Pro His His His Pro TrpGlu 245 250 255 cct tct gtg aga gag agc ctc tgt tcc aca tgg ccc tgg aaaaat gag 816 Pro Ser Val Arg Glu Ser Leu Cys Ser Thr Trp Pro Trp Lys AsnGlu 260 265 270 gat ttt aac caa aaa ttc aca cag ctg cta ctt cta caa agacct cac 864 Asp Phe Asn Gln Lys Phe Thr Gln Leu Leu Leu Leu Gln Arg ProHis 275 280 285 ccc aga agc caa gat ccc ctg gtc aag aga agc tgg cct gattat gtg 912 Pro Arg Ser Gln Asp Pro Leu Val Lys Arg Ser Trp Pro Asp TyrVal 290 295 300 gag gag aat cga gga cat tta att gag atc aga gac tta tttggc cca 960 Glu Glu Asn Arg Gly His Leu Ile Glu Ile Arg Asp Leu Phe GlyPro 305 310 315 320 ggc ctg gat acc caa gaa cct cgc ata gtc ata ctg cagggg gct gct 1008 Gly Leu Asp Thr Gln Glu Pro Arg Ile Val Ile Leu Gln GlyAla Ala 325 330 335 gga att ggg aag tca aca ctg gcc agg cag gtg aag gaagcc tgg ggg 1056 Gly Ile Gly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu AlaTrp Gly 340 345 350 aga ggc cag ctg tat ggg gac cgc ttc cag cat gtc ttctac ttc agc 1104 Arg Gly Gln Leu Tyr Gly Asp Arg Phe Gln His Val Phe TyrPhe Ser 355 360 365 tgc aga gag ctg gcc cag tcc aag gtg gtg agt ctc gctgag ctc atc 1152 Cys Arg Glu Leu Ala Gln Ser Lys Val Val Ser Leu Ala GluLeu Ile 370 375 380 gga aaa gat ggg aca gcc act ccg gct ccc att aga cagatc ctg tct 1200 Gly Lys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln IleLeu Ser 385 390 395 400 agg cca gag cgg ctg ctc ttc atc ctc gat ggt gtagat gag cca gga 1248 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val AspGlu Pro Gly 405 410 415 tgg gtc ttg cag gag ccg agt tct gag ctc tgt ctgcac tgg agc cag 1296 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu Cys Leu HisTrp Ser Gln 420 425 430 cca cag ccg gcg gat gca ctg ctg ggc agt ttg ctgggg aaa act ata 1344 Pro Gln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu GlyLys Thr Ile 435 440 445 ctt ccc gag gca tcc ttc ctg atc acg gct cgg accaca gct ctg cag 1392 Leu Pro Glu Ala Ser Phe Leu Ile Thr Ala Arg Thr ThrAla Leu Gln 450 455 460 aac ctc att cct tct ttg gag cag gca cgt tgg gtagag gtc ctg ggg 1440 Asn Leu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val GluVal Leu Gly 465 470 475 480 ttc tct gag tcc agc agg aag gaa tat ttc tacaga tat ttc aca gat 1488 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr ArgTyr Phe Thr Asp 485 490 495 gaa agg caa gca att aga gcc ttt agg ttg gtcaaa tca aac aaa gag 1536 Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val LysSer Asn Lys Glu 500 505 510 ctc tgg gcc ctg tgt ctt gtg ccc tgg gtg tcctgg ctg gcc tgc act 1584 Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser TrpLeu Ala Cys Thr 515 520 525 tgc ctg atg cag cag atg aag cgg aag gaa aaactc aca ctg act tcc 1632 Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys LeuThr Leu Thr Ser 530 535 540 aag acc acc aca acc ctc tgt cta cat tac cttgcc cag gct ctc caa 1680 Lys Thr Thr Thr Thr Leu Cys Leu His Tyr Leu AlaGln Ala Leu Gln 545 550 555 560 gct cag cca ttg gga ccc cag ctc aga gacctc tgc tct ctg gct gct 1728 Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp LeuCys Ser Leu Ala Ala 565 570 575 gag ggc atc tgg caa aaa aag acc ctt ttcagt cca gat gac ctc agg 1776 Glu Gly Ile Trp Gln Lys Lys Thr Leu Phe SerPro Asp Asp Leu Arg 580 585 590 aag cat ggg tta gat ggg gcc atc atc tccacc ttc ttg aag atg ggt 1824 Lys His Gly Leu Asp Gly Ala Ile Ile Ser ThrPhe Leu Lys Met Gly 595 600 605 att ctt caa gag cac ccc atc cct ctg agctac agc ttc att cac ctc 1872 Ile Leu Gln Glu His Pro Ile Pro Leu Ser TyrSer Phe Ile His Leu 610 615 620 tgt ttc caa gag ttc ttt gca gca atg tcctat gtc ttg gag gat gag 1920 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser TyrVal Leu Glu Asp Glu 625 630 635 640 aag ggg aga ggt aaa cat tct aat tgcatc ata gat ttg gaa aag acg 1968 Lys Gly Arg Gly Lys His Ser Asn Cys IleIle Asp Leu Glu Lys Thr 645 650 655 cta gaa gca tat gga ata cat ggc ctgttt ggg gca tca acc aca cgt 2016 Leu Glu Ala Tyr Gly Ile His Gly Leu PheGly Ala Ser Thr Thr Arg 660 665 670 ttc cta ttg ggc ctg tta agt gat gagggg gag aga gag atg gag aac 2064 Phe Leu Leu Gly Leu Leu Ser Asp Glu GlyGlu Arg Glu Met Glu Asn 675 680 685 atc ttt cac tgc cgg ctg tct cag gggagg aac ctg atg cag tgg gtc 2112 Ile Phe His Cys Arg Leu Ser Gln Gly ArgAsn Leu Met Gln Trp Val 690 695 700 ccg tcc ctg cag ctg ctg ctg cag ccacac tct ctg gag tcc ctc cac 2160 Pro Ser Leu Gln Leu Leu Leu Gln Pro HisSer Leu Glu Ser Leu His 705 710 715 720 tgc ttg tac gag act cgg aac aaaacg ttc ctg aca caa gtg atg gcc 2208 Cys Leu Tyr Glu Thr Arg Asn Lys ThrPhe Leu Thr Gln Val Met Ala 725 730 735 cat ttc gaa gaa atg ggc atg tgtgta gaa aca gac atg gag ctc tta 2256 His Phe Glu Glu Met Gly Met Cys ValGlu Thr Asp Met Glu Leu Leu 740 745 750 gtg tgc act ttc tgc att aaa ttcagc cgc cac gtg aag aag ctt cag 2304 Val Cys Thr Phe Cys Ile Lys Phe SerArg His Val Lys Lys Leu Gln 755 760 765 ctg att gag ggc agg cag cac agatca aca tgg agc ccc acc atg gta 2352 Leu Ile Glu Gly Arg Gln His Arg SerThr Trp Ser Pro Thr Met Val 770 775 780 gtc ctg ttc agg tgg gtc cca gtcaca gat gcc tat tgg cag att ctc 2400 Val Leu Phe Arg Trp Val Pro Val ThrAsp Ala Tyr Trp Gln Ile Leu 785 790 795 800 ttc tcc gtc ctc aag gtc accaga aac ctg aag gag ctg gac cta agt 2448 Phe Ser Val Leu Lys Val Thr ArgAsn Leu Lys Glu Leu Asp Leu Ser 805 810 815 gga aac tcg ctg agc cac tctgca gtg aag agt ctt tgt aag acc ctg 2496 Gly Asn Ser Leu Ser His Ser AlaVal Lys Ser Leu Cys Lys Thr Leu 820 825 830 aga cgc cct cgc tgc ctc ctggag acc ctg cgg ttg gct ggc tgt ggc 2544 Arg Arg Pro Arg Cys Leu Leu GluThr Leu Arg Leu Ala Gly Cys Gly 835 840 845 ctc aca gct gag gac tgc aaggac ctt gcc ttt ggg ctg aga gcc aac 2592 Leu Thr Ala Glu Asp Cys Lys AspLeu Ala Phe Gly Leu Arg Ala Asn 850 855 860 cag acc ctg acc gag ctg gacctg agc ttc aat gtg ctc acg gat gct 2640 Gln Thr Leu Thr Glu Leu Asp LeuSer Phe Asn Val Leu Thr Asp Ala 865 870 875 880 gga gcc aaa cac ctt tgccag aga ctg aga cag ccg agc tgc aag cta 2688 Gly Ala Lys His Leu Cys GlnArg Leu Arg Gln Pro Ser Cys Lys Leu 885 890 895 cag cga ctg cag ctg gtcagc tgt ggc ctc acg tct gac tgc tgc cag 2736 Gln Arg Leu Gln Leu Val SerCys Gly Leu Thr Ser Asp Cys Cys Gln 900 905 910 gac ctg gcc tct gtg cttagt gcc agc ccc agc ctg aag gag cta gac 2784 Asp Leu Ala Ser Val Leu SerAla Ser Pro Ser Leu Lys Glu Leu Asp 915 920 925 ctg cag cag aac aac ctggat gac gtt ggc gtg cga ctg ctc tgt gag 2832 Leu Gln Gln Asn Asn Leu AspAsp Val Gly Val Arg Leu Leu Cys Glu 930 935 940 ggg ctc agg cat cct gcctgc aaa ctc ata cgc ctg ggg aaa cca agt 2880 Gly Leu Arg His Pro Ala CysLys Leu Ile Arg Leu Gly Lys Pro Ser 945 950 955 960 gtg atg acc cct actgag ggc ctg gat acg gga gag atg agt aat agc 2928 Val Met Thr Pro Thr GluGly Leu Asp Thr Gly Glu Met Ser Asn Ser 965 970 975 aca tcc tca ctc aagcgg cag aga ctc gga tca gag agg gcg gct tcc 2976 Thr Ser Ser Leu Lys ArgGln Arg Leu Gly Ser Glu Arg Ala Ala Ser 980 985 990 cat gtt gct cag gctaat ctc aaa ctc ctg gac gtg agc aag atc ttc 3024 His Val Ala Gln Ala AsnLeu Lys Leu Leu Asp Val Ser Lys Ile Phe 995 1000 1005 cca att gct gagatt gca gag gaa agc tcc cca gag gta gta ccg gtg 3072 Pro Ile Ala Glu IleAla Glu Glu Ser Ser Pro Glu Val Val Pro Val 1010 1015 1020 gaa ctc ttgtgc gtg cct tct cct gcc tct caa ggg gac ctg cat acg 3120 Glu Leu Leu CysVal Pro Ser Pro Ala Ser Gln Gly Asp Leu His Thr 1025 1030 1035 1040 aagcct ttg ggg act gac gat gac ttc tgg ggc ccc acg ggg cct gtg 3168 Lys ProLeu Gly Thr Asp Asp Asp Phe Trp Gly Pro Thr Gly Pro Val 1045 1050 1055gct act gag gta gtt gac aaa gaa aag aac ttg tac cga gtt cac ttc 3216 AlaThr Glu Val Val Asp Lys Glu Lys Asn Leu Tyr Arg Val His Phe 1060 10651070 cct gta gct ggc tcc tac cgc tgg ccc aac acg ggt ctc tgc ttt gtg3264 Pro Val Ala Gly Ser Tyr Arg Trp Pro Asn Thr Gly Leu Cys Phe Val1075 1080 1085 atg aga gaa gcg gtg acc gtt gag att gaa ttc tgt gtg tgggac cag 3312 Met Arg Glu Ala Val Thr Val Glu Ile Glu Phe Cys Val Trp AspGln 1090 1095 1100 ttc ctg ggt gag atc aac cca cag cac agc tgg atg gtggca ggg cct 3360 Phe Leu Gly Glu Ile Asn Pro Gln His Ser Trp Met Val AlaGly Pro 1105 1110 1115 1120 ctg ctg gac atc aag gct gag cct gga gct gtggaa gct gtg cac ctc 3408 Leu Leu Asp Ile Lys Ala Glu Pro Gly Ala Val GluAla Val His Leu 1125 1130 1135 cct cac ttt gtg gct ctc caa ggg ggc catgtg gac aca tcc ctg ttc 3456 Pro His Phe Val Ala Leu Gln Gly Gly His ValAsp Thr Ser Leu Phe 1140 1145 1150 caa atg gcc cac ttt aaa gag gag gggatg ctc ctg gag aag cca gcc 3504 Gln Met Ala His Phe Lys Glu Glu Gly MetLeu Leu Glu Lys Pro Ala 1155 1160 1165 agg gtg gag ctg cat cac ata gttctg gaa aac ccc agc ttc tcc ccc 3552 Arg Val Glu Leu His His Ile Val LeuGlu Asn Pro Ser Phe Ser Pro 1170 1175 1180 ttg gga gtc ctc ctg aaa atgatc cat aat gcc ctg cgc ttc att ccc 3600 Leu Gly Val Leu Leu Lys Met IleHis Asn Ala Leu Arg Phe Ile Pro 1185 1190 1195 1200 gtc acc tct gtg gtgttg ctt tac cac cgc gtc cat cct gag gaa gtc 3648 Val Thr Ser Val Val LeuLeu Tyr His Arg Val His Pro Glu Glu Val 1205 1210 1215 acc ttc cac ctctac ctg atc cca agt gac tgc tcc att cgg aag gaa 3696 Thr Phe His Leu TyrLeu Ile Pro Ser Asp Cys Ser Ile Arg Lys Glu 1220 1225 1230 ctg gag ctctgc tat cga agc cct gga gaa gac cag ctg ttc tcg gag 3744 Leu Glu Leu CysTyr Arg Ser Pro Gly Glu Asp Gln Leu Phe Ser Glu 1235 1240 1245 ttc tacgtt ggc cac ttg gga tca ggg atc agg ctg caa gtg aaa gac 3792 Phe Tyr ValGly His Leu Gly Ser Gly Ile Arg Leu Gln Val Lys Asp 1250 1255 1260 aagaaa gat gag act ctg gtg tgg gag gcc ttg gtg aaa cca gga gat 3840 Lys LysAsp Glu Thr Leu Val Trp Glu Ala Leu Val Lys Pro Gly Asp 1265 1270 12751280 ctc atg cct gca act act ctg atc cct cca gcc cgc ata gcc gta cct3888 Leu Met Pro Ala Thr Thr Leu Ile Pro Pro Ala Arg Ile Ala Val Pro1285 1290 1295 tca cct ctg gat gcc ccg cag ttg ctg cac ttt gtg gac cagtat cga 3936 Ser Pro Leu Asp Ala Pro Gln Leu Leu His Phe Val Asp Gln TyrArg 1300 1305 1310 gag cag ctg ata gcc cga gtg aca tcg gtg gag gtt gtcttg gac aaa 3984 Glu Gln Leu Ile Ala Arg Val Thr Ser Val Glu Val Val LeuAsp Lys 1315 1320 1325 ctg cat gga cag gtg ctg agc cag gag cag tac gagagg gtg ctg gct 4032 Leu His Gly Gln Val Leu Ser Gln Glu Gln Tyr Glu ArgVal Leu Ala 1330 1335 1340 gag aac acg agg ccc agc cag atg cgg aag ctgttc agc ttg agc cag 4080 Glu Asn Thr Arg Pro Ser Gln Met Arg Lys Leu PheSer Leu Ser Gln 1345 1350 1355 1360 tcc tgg gac cgg aag tgc aaa gat ggactc tac caa gcc ctg aag gag 4128 Ser Trp Asp Arg Lys Cys Lys Asp Gly LeuTyr Gln Ala Leu Lys Glu 1365 1370 1375 acc cat cct cac ctc att atg gaactc tgg gag aag ggc agc aaa aag 4176 Thr His Pro His Leu Ile Met Glu LeuTrp Glu Lys Gly Ser Lys Lys 1380 1385 1390 gga ctc ctg cca ctc agc agctga 4200 Gly Leu Leu Pro Leu Ser Ser 1395 4 1399 PRT Homo sapiens 4 MetAla Gly Gly Ala Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15Lys Lys Glu Glu Leu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30His Ser Arg Ser Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45Ser Gly Met Glu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60Arg Ala Trp Asp Leu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 7580 Ser Leu Cys Ala Gln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 9095 Pro Tyr Ser Pro Ser Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100105 110 Ser Thr Ala Val Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys115 120 125 Thr Gln Gly Ser Glu Arg Arg Val Leu Arg Gln Leu Pro Asp ThrSer 130 135 140 Gly Arg Arg Trp Arg Glu Ile Ser Ala Ser Leu Leu Tyr GlnAla Leu 145 150 155 160 Pro Ser Ser Pro Asp His Glu Ser Pro Ser Gln GluSer Pro Asn Ala 165 170 175 Pro Thr Ser Thr Ala Val Leu Gly Ser Trp GlySer Pro Pro Gln Pro 180 185 190 Ser Leu Ala Pro Arg Glu Gln Glu Ala ProGly Thr Gln Trp Pro Leu 195 200 205 Asp Glu Thr Ser Gly Ile Tyr Tyr ThrGlu Ile Arg Glu Arg Glu Arg 210 215 220 Glu Lys Ser Glu Lys Gly Arg ProPro Trp Ala Ala Val Val Gly Thr 225 230 235 240 Pro Pro Gln Ala His ThrSer Leu Gln Pro His His His Pro Trp Glu 245 250 255 Pro Ser Val Arg GluSer Leu Cys Ser Thr Trp Pro Trp Lys Asn Glu 260 265 270 Asp Phe Asn GlnLys Phe Thr Gln Leu Leu Leu Leu Gln Arg Pro His 275 280 285 Pro Arg SerGln Asp Pro Leu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300 Glu GluAsn Arg Gly His Leu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320Gly Leu Asp Thr Gln Glu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330335 Gly Ile Gly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340345 350 Arg Gly Gln Leu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser355 360 365 Cys Arg Glu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu LeuIle 370 375 380 Gly Lys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln IleLeu Ser 385 390 395 400 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly ValAsp Glu Pro Gly 405 410 415 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu CysLeu His Trp Ser Gln 420 425 430 Pro Gln Pro Ala Asp Ala Leu Leu Gly SerLeu Leu Gly Lys Thr Ile 435 440 445 Leu Pro Glu Ala Ser Phe Leu Ile ThrAla Arg Thr Thr Ala Leu Gln 450 455 460 Asn Leu Ile Pro Ser Leu Glu GlnAla Arg Trp Val Glu Val Leu Gly 465 470 475 480 Phe Ser Glu Ser Ser ArgLys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485 490 495 Glu Arg Gln Ala IleArg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu 500 505 510 Leu Trp Ala LeuCys Leu Val Pro Trp Val Ser Trp Leu Ala Cys Thr 515 520 525 Cys Leu MetGln Gln Met Lys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535 540 Lys ThrThr Thr Thr Leu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570575 Glu Gly Ile Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580585 590 Lys His Gly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly595 600 605 Ile Leu Gln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile HisLeu 610 615 620 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu GluAsp Glu 625 630 635 640 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile AspLeu Glu Lys Thr 645 650 655 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe GlyAla Ser Thr Thr Arg 660 665 670 Phe Leu Leu Gly Leu Leu Ser Asp Glu GlyGlu Arg Glu Met Glu Asn 675 680 685 Ile Phe His Cys Arg Leu Ser Gln GlyArg Asn Leu Met Gln Trp Val 690 695 700 Pro Ser Leu Gln Leu Leu Leu GlnPro His Ser Leu Glu Ser Leu His 705 710 715 720 Cys Leu Tyr Glu Thr ArgAsn Lys Thr Phe Leu Thr Gln Val Met Ala 725 730 735 His Phe Glu Glu MetGly Met Cys Val Glu Thr Asp Met Glu Leu Leu 740 745 750 Val Cys Thr PheCys Ile Lys Phe Ser Arg His Val Lys Lys Leu Gln 755 760 765 Leu Ile GluGly Arg Gln His Arg Ser Thr Trp Ser Pro Thr Met Val 770 775 780 Val LeuPhe Arg Trp Val Pro Val Thr Asp Ala Tyr Trp Gln Ile Leu 785 790 795 800Phe Ser Val Leu Lys Val Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805 810815 Gly Asn Ser Leu Ser His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820825 830 Arg Arg Pro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly835 840 845 Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg AlaAsn 850 855 860 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu ThrAsp Ala 865 870 875 880 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln ProSer Cys Lys Leu 885 890 895 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu ThrSer Asp Cys Cys Gln 900 905 910 Asp Leu Ala Ser Val Leu Ser Ala Ser ProSer Leu Lys Glu Leu Asp 915 920 925 Leu Gln Gln Asn Asn Leu Asp Asp ValGly Val Arg Leu Leu Cys Glu 930 935 940 Gly Leu Arg His Pro Ala Cys LysLeu Ile Arg Leu Gly Lys Pro Ser 945 950 955 960 Val Met Thr Pro Thr GluGly Leu Asp Thr Gly Glu Met Ser Asn Ser 965 970 975 Thr Ser Ser Leu LysArg Gln Arg Leu Gly Ser Glu Arg Ala Ala Ser 980 985 990 His Val Ala GlnAla Asn Leu Lys Leu Leu Asp Val Ser Lys Ile Phe 995 1000 1005 Pro IleAla Glu Ile Ala Glu Glu Ser Ser Pro Glu Val Val Pro Val 1010 1015 1020Glu Leu Leu Cys Val Pro Ser Pro Ala Ser Gln Gly Asp Leu His Thr 10251030 1035 1040 Lys Pro Leu Gly Thr Asp Asp Asp Phe Trp Gly Pro Thr GlyPro Val 1045 1050 1055 Ala Thr Glu Val Val Asp Lys Glu Lys Asn Leu TyrArg Val His Phe 1060 1065 1070 Pro Val Ala Gly Ser Tyr Arg Trp Pro AsnThr Gly Leu Cys Phe Val 1075 1080 1085 Met Arg Glu Ala Val Thr Val GluIle Glu Phe Cys Val Trp Asp Gln 1090 1095 1100 Phe Leu Gly Glu Ile AsnPro Gln His Ser Trp Met Val Ala Gly Pro 1105 1110 1115 1120 Leu Leu AspIle Lys Ala Glu Pro Gly Ala Val Glu Ala Val His Leu 1125 1130 1135 ProHis Phe Val Ala Leu Gln Gly Gly His Val Asp Thr Ser Leu Phe 1140 11451150 Gln Met Ala His Phe Lys Glu Glu Gly Met Leu Leu Glu Lys Pro Ala1155 1160 1165 Arg Val Glu Leu His His Ile Val Leu Glu Asn Pro Ser PheSer Pro 1170 1175 1180 Leu Gly Val Leu Leu Lys Met Ile His Asn Ala LeuArg Phe Ile Pro 1185 1190 1195 1200 Val Thr Ser Val Val Leu Leu Tyr HisArg Val His Pro Glu Glu Val 1205 1210 1215 Thr Phe His Leu Tyr Leu IlePro Ser Asp Cys Ser Ile Arg Lys Glu 1220 1225 1230 Leu Glu Leu Cys TyrArg Ser Pro Gly Glu Asp Gln Leu Phe Ser Glu 1235 1240 1245 Phe Tyr ValGly His Leu Gly Ser Gly Ile Arg Leu Gln Val Lys Asp 1250 1255 1260 LysLys Asp Glu Thr Leu Val Trp Glu Ala Leu Val Lys Pro Gly Asp 1265 12701275 1280 Leu Met Pro Ala Thr Thr Leu Ile Pro Pro Ala Arg Ile Ala ValPro 1285 1290 1295 Ser Pro Leu Asp Ala Pro Gln Leu Leu His Phe Val AspGln Tyr Arg 1300 1305 1310 Glu Gln Leu Ile Ala Arg Val Thr Ser Val GluVal Val Leu Asp Lys 1315 1320 1325 Leu His Gly Gln Val Leu Ser Gln GluGln Tyr Glu Arg Val Leu Ala 1330 1335 1340 Glu Asn Thr Arg Pro Ser GlnMet Arg Lys Leu Phe Ser Leu Ser Gln 1345 1350 1355 1360 Ser Trp Asp ArgLys Cys Lys Asp Gly Leu Tyr Gln Ala Leu Lys Glu 1365 1370 1375 Thr HisPro His Leu Ile Met Glu Leu Trp Glu Lys Gly Ser Lys Lys 1380 1385 1390Gly Leu Leu Pro Leu Ser Ser 1395 5 4332 DNA Homo sapiens CDS (1)..(4332)5 atg gct ggc gga gcc tgg ggc cgc ctg gcc tgt tac ttg gag ttc ctg 48 MetAla Gly Gly Ala Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15aag aag gag gag ctg aag gag ttc cag ctt ctg ctc gcc aat aaa gcg 96 LysLys Glu Glu Leu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 cactcc agg agc tct tcg ggt gag aca ccc gct cag cca gag aag acg 144 His SerArg Ser Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45 agt ggcatg gag gtg gcc tcg tac ctg gtg gct cag tat ggg gag cag 192 Ser Gly MetGlu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 cgg gcc tgggac cta gcc ctc cat acc tgg gag cag atg ggg ctg agg 240 Arg Ala Trp AspLeu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80 tca ctg tgcgcc caa gcc cag gaa ggg gca ggc cac tct ccc tca ttc 288 Ser Leu Cys AlaGln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 90 95 ccc tac agc ccaagt gaa ccc cac ctg ggg tct ccc agc caa ccc acc 336 Pro Tyr Ser Pro SerGlu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100 105 110 tcc acc gca gtgcta atg ccc tgg atc cat gaa ttg ccg gcg ggg tgc 384 Ser Thr Ala Val LeuMet Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115 120 125 acc cag ggc tcagag aga agg gtt ttg aga cag ctg cct gac aca tct 432 Thr Gln Gly Ser GluArg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser 130 135 140 gga cgc cgc tggaga gaa atc tct gcc tca ctc ctc tac caa gct ctt 480 Gly Arg Arg Trp ArgGlu Ile Ser Ala Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 cca agc tcccca gac cat gag tct cca agc cag gag tca ccc aac gcc 528 Pro Ser Ser ProAsp His Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 ccc aca tccaca gca gtg ctg ggg agc tgg gga tcc cca cct cag ccc 576 Pro Thr Ser ThrAla Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 agc cta gcaccc aga gag cag gag gct cct ggg acc caa tgg cct ctg 624 Ser Leu Ala ProArg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205 gat gaa acgtca gga att tac tac aca gaa atc aga gaa aga gag aga 672 Asp Glu Thr SerGly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215 220 gag aaa tcagag aaa ggc agg ccc cca tgg gca gcg gtg gta gga acg 720 Glu Lys Ser GluLys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr 225 230 235 240 ccc ccacag gcg cac acc agc cta cag ccc cac cac cac cca tgg gag 768 Pro Pro GlnAla His Thr Ser Leu Gln Pro His His His Pro Trp Glu 245 250 255 cct tctgtg aga gag agc ctc tgt tcc aca tgg ccc tgg aaa aat gag 816 Pro Ser ValArg Glu Ser Leu Cys Ser Thr Trp Pro Trp Lys Asn Glu 260 265 270 gat tttaac caa aaa ttc aca cag ctg cta ctt cta caa aga cct cac 864 Asp Phe AsnGln Lys Phe Thr Gln Leu Leu Leu Leu Gln Arg Pro His 275 280 285 ccc agaagc caa gat ccc ctg gtc aag aga agc tgg cct gat tat gtg 912 Pro Arg SerGln Asp Pro Leu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300 gag gagaat cga gga cat tta att gag atc aga gac tta ttt ggc cca 960 Glu Glu AsnArg Gly His Leu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320 ggcctg gat acc caa gaa cct cgc ata gtc ata ctg cag ggg gct gct 1008 Gly LeuAsp Thr Gln Glu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330 335 ggaatt ggg aag tca aca ctg gcc agg cag gtg aag gaa gcc tgg ggg 1056 Gly IleGly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345 350 agaggc cag ctg tat ggg gac cgc ttc cag cat gtc ttc tac ttc agc 1104 Arg GlyGln Leu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355 360 365 tgcaga gag ctg gcc cag tcc aag gtg gtg agt ctc gct gag ctc atc 1152 Cys ArgGlu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu Leu Ile 370 375 380 ggaaaa gat ggg aca gcc act ccg gct ccc att aga cag atc ctg tct 1200 Gly LysAsp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395 400agg cca gag cgg ctg ctc ttc atc ctc gat ggt gta gat gag cca gga 1248 ArgPro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405 410 415tgg gtc ttg cag gag ccg agt tct gag ctc tgt ctg cac tgg agc cag 1296 TrpVal Leu Gln Glu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420 425 430cca cag ccg gcg gat gca ctg ctg ggc agt ttg ctg ggg aaa act ata 1344 ProGln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445ctt ccc gag gca tcc ttc ctg atc acg gct cgg acc aca gct ctg cag 1392 LeuPro Glu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450 455 460aac ctc att cct tct ttg gag cag gca cgt tgg gta gag gtc ctg ggg 1440 AsnLeu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465 470 475480 ttc tct gag tcc agc agg aag gaa tat ttc tac aga tat ttc aca gat 1488Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485 490495 gaa agg caa gca att aga gcc ttt agg ttg gtc aaa tca aac aaa gag 1536Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu 500 505510 ctc tgg gcc ctg tgt ctt gtg ccc tgg gtg tcc tgg ctg gcc tgc act 1584Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser Trp Leu Ala Cys Thr 515 520525 tgc ctg atg cag cag atg aag cgg aag gaa aaa ctc aca ctg act tcc 1632Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535540 aag acc acc aca acc ctc tgt cta cat tac ctt gcc cag gct ctc caa 1680Lys Thr Thr Thr Thr Leu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550555 560 gct cag cca ttg gga ccc cag ctc aga gac ctc tgc tct ctg gct gct1728 Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565570 575 gag ggc atc tgg caa aaa aag acc ctt ttc agt cca gat gac ctc agg1776 Glu Gly Ile Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580585 590 aag cat ggg tta gat ggg gcc atc atc tcc acc ttc ttg aag atg ggt1824 Lys His Gly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595600 605 att ctt caa gag cac ccc atc cct ctg agc tac agc ttc att cac ctc1872 Ile Leu Gln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu 610615 620 tgt ttc caa gag ttc ttt gca gca atg tcc tat gtc ttg gag gat gag1920 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625630 635 640 aag ggg aga ggt aaa cat tct aat tgc atc ata gat ttg gaa aagacg 1968 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr645 650 655 cta gaa gca tat gga ata cat ggc ctg ttt ggg gca tca acc acacgt 2016 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg660 665 670 ttc cta ttg ggc ctg tta agt gat gag ggg gag aga gag atg gagaac 2064 Phe Leu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn675 680 685 atc ttt cac tgc cgg ctg tct cag ggg agg aac ctg atg cag tgggtc 2112 Ile Phe His Cys Arg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val690 695 700 ccg tcc ctg cag ctg ctg ctg cag cca cac tct ctg gag tcc ctccac 2160 Pro Ser Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His705 710 715 720 tgc ttg tac gag act cgg aac aaa acg ttc ctg aca caa gtgatg gcc 2208 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val MetAla 725 730 735 cat ttc gaa gaa atg ggc atg tgt gta gaa aca gac atg gagctc tta 2256 His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp Met Glu LeuLeu 740 745 750 gtg tgc act ttc tgc att aaa ttc agc cgc cac gtg aag aagctt cag 2304 Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val Lys Lys LeuGln 755 760 765 ctg att gag ggc agg cag cac aga tca aca tgg agc ccc accatg gta 2352 Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp Ser Pro Thr MetVal 770 775 780 gtc ctg ttc agg tgg gtc cca gtc aca gat gcc tat tgg cagatt ctc 2400 Val Leu Phe Arg Trp Val Pro Val Thr Asp Ala Tyr Trp Gln IleLeu 785 790 795 800 ttc tcc gtc ctc aag gtc acc aga aac ctg aag gag ctggac cta agt 2448 Phe Ser Val Leu Lys Val Thr Arg Asn Leu Lys Glu Leu AspLeu Ser 805 810 815 gga aac tcg ctg agc cac tct gca gtg aag agt ctt tgtaag acc ctg 2496 Gly Asn Ser Leu Ser His Ser Ala Val Lys Ser Leu Cys LysThr Leu 820 825 830 aga cgc cct cgc tgc ctc ctg gag acc ctg cgg ttg gctggc tgt ggc 2544 Arg Arg Pro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala GlyCys Gly 835 840 845 ctc aca gct gag gac tgc aag gac ctt gcc ttt ggg ctgaga gcc aac 2592 Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu ArgAla Asn 850 855 860 cag acc ctg acc gag ctg gac ctg agc ttc aat gtg ctcacg gat gct 2640 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu ThrAsp Ala 865 870 875 880 gga gcc aaa cac ctt tgc cag aga ctg aga cag ccgagc tgc aag cta 2688 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln Pro SerCys Lys Leu 885 890 895 cag cga ctg cag ctg gtc agc tgt ggc ctc acg tctgac tgc tgc cag 2736 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu Thr Ser AspCys Cys Gln 900 905 910 gac ctg gcc tct gtg ctt agt gcc agc ccc agc ctgaag gag cta gac 2784 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu LysGlu Leu Asp 915 920 925 ctg cag cag aac aac ctg gat gac gtt ggc gtg cgactg ctc tgt gag 2832 Leu Gln Gln Asn Asn Leu Asp Asp Val Gly Val Arg LeuLeu Cys Glu 930 935 940 ggg ctc agg cat cct gcc tgc aaa ctc ata cgc ctgggg aaa cca agt 2880 Gly Leu Arg His Pro Ala Cys Lys Leu Ile Arg Leu GlyLys Pro Ser 945 950 955 960 gtg atg acc cct act gag ggc ctg gat acg ggagag atg agt aat agc 2928 Val Met Thr Pro Thr Glu Gly Leu Asp Thr Gly GluMet Ser Asn Ser 965 970 975 aca tcc tca ctc aag cgg cag aga ctc gga tcagag agg gcg gct tcc 2976 Thr Ser Ser Leu Lys Arg Gln Arg Leu Gly Ser GluArg Ala Ala Ser 980 985 990 cat gtt gct cag gct aat ctc aaa ctc ctg gacgtg agc aag atc ttc 3024 His Val Ala Gln Ala Asn Leu Lys Leu Leu Asp ValSer Lys Ile Phe 995 1000 1005 cca att gct gag att gca gag gaa agc tcccca gag gta gta ccg gtg 3072 Pro Ile Ala Glu Ile Ala Glu Glu Ser Ser ProGlu Val Val Pro Val 1010 1015 1020 gaa ctc ttg tgc gtg cct tct cct gcctct caa ggg gac ctg cat acg 3120 Glu Leu Leu Cys Val Pro Ser Pro Ala SerGln Gly Asp Leu His Thr 1025 1030 1035 1040 aag cct ttg ggg act gac gatgac ttc tgg ggc ccc acg ggg cct gtg 3168 Lys Pro Leu Gly Thr Asp Asp AspPhe Trp Gly Pro Thr Gly Pro Val 1045 1050 1055 gct act gag gta gtt gacaaa gaa aag aac ttg tac cga gtt cac ttc 3216 Ala Thr Glu Val Val Asp LysGlu Lys Asn Leu Tyr Arg Val His Phe 1060 1065 1070 cct gta gct ggc tcctac cgc tgg ccc aac acg ggt ctc tgc ttt gtg 3264 Pro Val Ala Gly Ser TyrArg Trp Pro Asn Thr Gly Leu Cys Phe Val 1075 1080 1085 atg aga gaa gcggtg acc gtt gag att gaa ttc tgt gtg tgg gac cag 3312 Met Arg Glu Ala ValThr Val Glu Ile Glu Phe Cys Val Trp Asp Gln 1090 1095 1100 ttc ctg ggtgag atc aac cca cag cac agc tgg atg gtg gca ggg cct 3360 Phe Leu Gly GluIle Asn Pro Gln His Ser Trp Met Val Ala Gly Pro 1105 1110 1115 1120 ctgctg gac atc aag gct gag cct gga gct gtg gaa gct gtg cac ctc 3408 Leu LeuAsp Ile Lys Ala Glu Pro Gly Ala Val Glu Ala Val His Leu 1125 1130 1135cct cac ttt gtg gct ctc caa ggg ggc cat gtg gac aca tcc ctg ttc 3456 ProHis Phe Val Ala Leu Gln Gly Gly His Val Asp Thr Ser Leu Phe 1140 11451150 caa atg gcc cac ttt aaa gag gag ggg atg ctc ctg gag aag cca gcc3504 Gln Met Ala His Phe Lys Glu Glu Gly Met Leu Leu Glu Lys Pro Ala1155 1160 1165 agg gtg gag ctg cat cac ata gtt ctg gaa aac ccc agc ttctcc ccc 3552 Arg Val Glu Leu His His Ile Val Leu Glu Asn Pro Ser Phe SerPro 1170 1175 1180 ttg gga gtc ctc ctg aaa atg atc cat aat gcc ctg cgcttc att ccc 3600 Leu Gly Val Leu Leu Lys Met Ile His Asn Ala Leu Arg PheIle Pro 1185 1190 1195 1200 gtc acc tct gtg gtg ttg ctt tac cac cgc gtccat cct gag gaa gtc 3648 Val Thr Ser Val Val Leu Leu Tyr His Arg Val HisPro Glu Glu Val 1205 1210 1215 acc ttc cac ctc tac ctg atc cca agt gactgc tcc att cgg aag gcc 3696 Thr Phe His Leu Tyr Leu Ile Pro Ser Asp CysSer Ile Arg Lys Ala 1220 1225 1230 ata gat gat cta gaa atg aaa ttc cagttt gtg cga atc cac aag cca 3744 Ile Asp Asp Leu Glu Met Lys Phe Gln PheVal Arg Ile His Lys Pro 1235 1240 1245 ccc ccg ctg acc cca ctt tat atgggc tgt cgt tac act gtg tct ggg 3792 Pro Pro Leu Thr Pro Leu Tyr Met GlyCys Arg Tyr Thr Val Ser Gly 1250 1255 1260 tct ggt tca ggg atg ctg gaaata ctc ccc aag gaa ctg gag ctc tgc 3840 Ser Gly Ser Gly Met Leu Glu IleLeu Pro Lys Glu Leu Glu Leu Cys 1265 1270 1275 1280 tat cga agc cct ggagaa gac cag ctg ttc tcg gag ttc tac gtt ggc 3888 Tyr Arg Ser Pro Gly GluAsp Gln Leu Phe Ser Glu Phe Tyr Val Gly 1285 1290 1295 cac ttg gga tcaggg atc agg ctg caa gtg aaa gac aag aaa gat gag 3936 His Leu Gly Ser GlyIle Arg Leu Gln Val Lys Asp Lys Lys Asp Glu 1300 1305 1310 act ctg gtgtgg gag gcc ttg gtg aaa cca gga gat ctc atg cct gca 3984 Thr Leu Val TrpGlu Ala Leu Val Lys Pro Gly Asp Leu Met Pro Ala 1315 1320 1325 act actctg atc cct cca gcc cgc ata gcc gta cct tca cct ctg gat 4032 Thr Thr LeuIle Pro Pro Ala Arg Ile Ala Val Pro Ser Pro Leu Asp 1330 1335 1340 gccccg cag ttg ctg cac ttt gtg gac cag tat cga gag cag ctg ata 4080 Ala ProGln Leu Leu His Phe Val Asp Gln Tyr Arg Glu Gln Leu Ile 1345 1350 13551360 gcc cga gtg aca tcg gtg gag gtt gtc ttg gac aaa ctg cat gga cag4128 Ala Arg Val Thr Ser Val Glu Val Val Leu Asp Lys Leu His Gly Gln1365 1370 1375 gtg ctg agc cag gag cag tac gag agg gtg ctg gct gag aacacg agg 4176 Val Leu Ser Gln Glu Gln Tyr Glu Arg Val Leu Ala Glu Asn ThrArg 1380 1385 1390 ccc agc cag atg cgg aag ctg ttc agc ttg agc cag tcctgg gac cgg 4224 Pro Ser Gln Met Arg Lys Leu Phe Ser Leu Ser Gln Ser TrpAsp Arg 1395 1400 1405 aag tgc aaa gat gga ctc tac caa gcc ctg aag gagacc cat cct cac 4272 Lys Cys Lys Asp Gly Leu Tyr Gln Ala Leu Lys Glu ThrHis Pro His 1410 1415 1420 ctc att atg gaa ctc tgg gag aag ggc agc aaaaag gga ctc ctg cca 4320 Leu Ile Met Glu Leu Trp Glu Lys Gly Ser Lys LysGly Leu Leu Pro 1425 1430 1435 1440 ctc agc agc tga 4332 Leu Ser Ser 61443 PRT Homo sapiens 6 Met Ala Gly Gly Ala Trp Gly Arg Leu Ala Cys TyrLeu Glu Phe Leu 1 5 10 15 Lys Lys Glu Glu Leu Lys Glu Phe Gln Leu LeuLeu Ala Asn Lys Ala 20 25 30 His Ser Arg Ser Ser Ser Gly Glu Thr Pro AlaGln Pro Glu Lys Thr 35 40 45 Ser Gly Met Glu Val Ala Ser Tyr Leu Val AlaGln Tyr Gly Glu Gln 50 55 60 Arg Ala Trp Asp Leu Ala Leu His Thr Trp GluGln Met Gly Leu Arg 65 70 75 80 Ser Leu Cys Ala Gln Ala Gln Glu Gly AlaGly His Ser Pro Ser Phe 85 90 95 Pro Tyr Ser Pro Ser Glu Pro His Leu GlySer Pro Ser Gln Pro Thr 100 105 110 Ser Thr Ala Val Leu Met Pro Trp IleHis Glu Leu Pro Ala Gly Cys 115 120 125 Thr Gln Gly Ser Glu Arg Arg ValLeu Arg Gln Leu Pro Asp Thr Ser 130 135 140 Gly Arg Arg Trp Arg Glu IleSer Ala Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 Pro Ser Ser Pro AspHis Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 Pro Thr Ser ThrAla Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 Ser Leu AlaPro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205 Asp GluThr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215 220 GluLys Ser Glu Lys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr 225 230 235240 Pro Pro Gln Ala His Thr Ser Leu Gln Pro His His His Pro Trp Glu 245250 255 Pro Ser Val Arg Glu Ser Leu Cys Ser Thr Trp Pro Trp Lys Asn Glu260 265 270 Asp Phe Asn Gln Lys Phe Thr Gln Leu Leu Leu Leu Gln Arg ProHis 275 280 285 Pro Arg Ser Gln Asp Pro Leu Val Lys Arg Ser Trp Pro AspTyr Val 290 295 300 Glu Glu Asn Arg Gly His Leu Ile Glu Ile Arg Asp LeuPhe Gly Pro 305 310 315 320 Gly Leu Asp Thr Gln Glu Pro Arg Ile Val IleLeu Gln Gly Ala Ala 325 330 335 Gly Ile Gly Lys Ser Thr Leu Ala Arg GlnVal Lys Glu Ala Trp Gly 340 345 350 Arg Gly Gln Leu Tyr Gly Asp Arg PheGln His Val Phe Tyr Phe Ser 355 360 365 Cys Arg Glu Leu Ala Gln Ser LysVal Val Ser Leu Ala Glu Leu Ile 370 375 380 Gly Lys Asp Gly Thr Ala ThrPro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395 400 Arg Pro Glu Arg LeuLeu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405 410 415 Trp Val Leu GlnGlu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420 425 430 Pro Gln ProAla Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445 Leu ProGlu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450 455 460 AsnLeu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465 470 475480 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485490 495 Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu500 505 510 Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser Trp Leu Ala CysThr 515 520 525 Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys Leu Thr LeuThr Ser 530 535 540 Lys Thr Thr Thr Thr Leu Cys Leu His Tyr Leu Ala GlnAla Leu Gln 545 550 555 560 Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp LeuCys Ser Leu Ala Ala 565 570 575 Glu Gly Ile Trp Gln Lys Lys Thr Leu PheSer Pro Asp Asp Leu Arg 580 585 590 Lys His Gly Leu Asp Gly Ala Ile IleSer Thr Phe Leu Lys Met Gly 595 600 605 Ile Leu Gln Glu His Pro Ile ProLeu Ser Tyr Ser Phe Ile His Leu 610 615 620 Cys Phe Gln Glu Phe Phe AlaAla Met Ser Tyr Val Leu Glu Asp Glu 625 630 635 640 Lys Gly Arg Gly LysHis Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645 650 655 Leu Glu Ala TyrGly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg 660 665 670 Phe Leu LeuGly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn 675 680 685 Ile PheHis Cys Arg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val 690 695 700 ProSer Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His 705 710 715720 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val Met Ala 725730 735 His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp Met Glu Leu Leu740 745 750 Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val Lys Lys LeuGln 755 760 765 Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp Ser Pro ThrMet Val 770 775 780 Val Leu Phe Arg Trp Val Pro Val Thr Asp Ala Tyr TrpGln Ile Leu 785 790 795 800 Phe Ser Val Leu Lys Val Thr Arg Asn Leu LysGlu Leu Asp Leu Ser 805 810 815 Gly Asn Ser Leu Ser His Ser Ala Val LysSer Leu Cys Lys Thr Leu 820 825 830 Arg Arg Pro Arg Cys Leu Leu Glu ThrLeu Arg Leu Ala Gly Cys Gly 835 840 845 Leu Thr Ala Glu Asp Cys Lys AspLeu Ala Phe Gly Leu Arg Ala Asn 850 855 860 Gln Thr Leu Thr Glu Leu AspLeu Ser Phe Asn Val Leu Thr Asp Ala 865 870 875 880 Gly Ala Lys His LeuCys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu 885 890 895 Gln Arg Leu GlnLeu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln 900 905 910 Asp Leu AlaSer Val Leu Ser Ala Ser Pro Ser Leu Lys Glu Leu Asp 915 920 925 Leu GlnGln Asn Asn Leu Asp Asp Val Gly Val Arg Leu Leu Cys Glu 930 935 940 GlyLeu Arg His Pro Ala Cys Lys Leu Ile Arg Leu Gly Lys Pro Ser 945 950 955960 Val Met Thr Pro Thr Glu Gly Leu Asp Thr Gly Glu Met Ser Asn Ser 965970 975 Thr Ser Ser Leu Lys Arg Gln Arg Leu Gly Ser Glu Arg Ala Ala Ser980 985 990 His Val Ala Gln Ala Asn Leu Lys Leu Leu Asp Val Ser Lys IlePhe 995 1000 1005 Pro Ile Ala Glu Ile Ala Glu Glu Ser Ser Pro Glu ValVal Pro Val 1010 1015 1020 Glu Leu Leu Cys Val Pro Ser Pro Ala Ser GlnGly Asp Leu His Thr 1025 1030 1035 1040 Lys Pro Leu Gly Thr Asp Asp AspPhe Trp Gly Pro Thr Gly Pro Val 1045 1050 1055 Ala Thr Glu Val Val AspLys Glu Lys Asn Leu Tyr Arg Val His Phe 1060 1065 1070 Pro Val Ala GlySer Tyr Arg Trp Pro Asn Thr Gly Leu Cys Phe Val 1075 1080 1085 Met ArgGlu Ala Val Thr Val Glu Ile Glu Phe Cys Val Trp Asp Gln 1090 1095 1100Phe Leu Gly Glu Ile Asn Pro Gln His Ser Trp Met Val Ala Gly Pro 11051110 1115 1120 Leu Leu Asp Ile Lys Ala Glu Pro Gly Ala Val Glu Ala ValHis Leu 1125 1130 1135 Pro His Phe Val Ala Leu Gln Gly Gly His Val AspThr Ser Leu Phe 1140 1145 1150 Gln Met Ala His Phe Lys Glu Glu Gly MetLeu Leu Glu Lys Pro Ala 1155 1160 1165 Arg Val Glu Leu His His Ile ValLeu Glu Asn Pro Ser Phe Ser Pro 1170 1175 1180 Leu Gly Val Leu Leu LysMet Ile His Asn Ala Leu Arg Phe Ile Pro 1185 1190 1195 1200 Val Thr SerVal Val Leu Leu Tyr His Arg Val His Pro Glu Glu Val 1205 1210 1215 ThrPhe His Leu Tyr Leu Ile Pro Ser Asp Cys Ser Ile Arg Lys Ala 1220 12251230 Ile Asp Asp Leu Glu Met Lys Phe Gln Phe Val Arg Ile His Lys Pro1235 1240 1245 Pro Pro Leu Thr Pro Leu Tyr Met Gly Cys Arg Tyr Thr ValSer Gly 1250 1255 1260 Ser Gly Ser Gly Met Leu Glu Ile Leu Pro Lys GluLeu Glu Leu Cys 1265 1270 1275 1280 Tyr Arg Ser Pro Gly Glu Asp Gln LeuPhe Ser Glu Phe Tyr Val Gly 1285 1290 1295 His Leu Gly Ser Gly Ile ArgLeu Gln Val Lys Asp Lys Lys Asp Glu 1300 1305 1310 Thr Leu Val Trp GluAla Leu Val Lys Pro Gly Asp Leu Met Pro Ala 1315 1320 1325 Thr Thr LeuIle Pro Pro Ala Arg Ile Ala Val Pro Ser Pro Leu Asp 1330 1335 1340 AlaPro Gln Leu Leu His Phe Val Asp Gln Tyr Arg Glu Gln Leu Ile 1345 13501355 1360 Ala Arg Val Thr Ser Val Glu Val Val Leu Asp Lys Leu His GlyGln 1365 1370 1375 Val Leu Ser Gln Glu Gln Tyr Glu Arg Val Leu Ala GluAsn Thr Arg 1380 1385 1390 Pro Ser Gln Met Arg Lys Leu Phe Ser Leu SerGln Ser Trp Asp Arg 1395 1400 1405 Lys Cys Lys Asp Gly Leu Tyr Gln AlaLeu Lys Glu Thr His Pro His 1410 1415 1420 Leu Ile Met Glu Leu Trp GluLys Gly Ser Lys Lys Gly Leu Leu Pro 1425 1430 1435 1440 Leu Ser Ser 71487 DNA Homo sapiens CDS (1)..(1296) 7 atg atg aga cag agg cag agc cattat tgt tcc gtg ctg ttc ctg agt 48 Met Met Arg Gln Arg Gln Ser His TyrCys Ser Val Leu Phe Leu Ser 1 5 10 15 gtc aac tat ctg ggg ggg aca ttccca gga gac att tgc tca gaa gag 96 Val Asn Tyr Leu Gly Gly Thr Phe ProGly Asp Ile Cys Ser Glu Glu 20 25 30 aat caa ata gtt tcc tct tat gct tctaaa gtc tgt ttt gag atc gaa 144 Asn Gln Ile Val Ser Ser Tyr Ala Ser LysVal Cys Phe Glu Ile Glu 35 40 45 gaa gat tat aaa aat cgt cag ttt ctg gggcct gaa gga aat gtg gat 192 Glu Asp Tyr Lys Asn Arg Gln Phe Leu Gly ProGlu Gly Asn Val Asp 50 55 60 gtt gag ttg att gat aag agc aca aac aga tacagc gtt tgg ttc ccc 240 Val Glu Leu Ile Asp Lys Ser Thr Asn Arg Tyr SerVal Trp Phe Pro 65 70 75 80 act gct ggc tgg tat ctg tgg tca gcc aca ggcctc ggc ttc ctg gta 288 Thr Ala Gly Trp Tyr Leu Trp Ser Ala Thr Gly LeuGly Phe Leu Val 85 90 95 agg gat gag gtc aca gtg acg att gcg ttt ggt tcctgg agt cag cac 336 Arg Asp Glu Val Thr Val Thr Ile Ala Phe Gly Ser TrpSer Gln His 100 105 110 ctg gcc ctg gac ctg cag cac cat gaa cag tgg ctggtg ggc ggc ccc 384 Leu Ala Leu Asp Leu Gln His His Glu Gln Trp Leu ValGly Gly Pro 115 120 125 ttg ttt gat gtc act gca gag cca gag gag gct gtcgcc gaa atc cac 432 Leu Phe Asp Val Thr Ala Glu Pro Glu Glu Ala Val AlaGlu Ile His 130 135 140 ctc ccc cac ttc atc tcc ctc caa ggt gag gtg gacgtc tcc tgg ttt 480 Leu Pro His Phe Ile Ser Leu Gln Gly Glu Val Asp ValSer Trp Phe 145 150 155 160 ctc gtt gcc cat ttt aag aat gaa ggg atg gtcctg gag cat cca gcc 528 Leu Val Ala His Phe Lys Asn Glu Gly Met Val LeuGlu His Pro Ala 165 170 175 cgg gtg gag cct ttc tat gct gtc ctg gaa agcccc agc ttc tct ctg 576 Arg Val Glu Pro Phe Tyr Ala Val Leu Glu Ser ProSer Phe Ser Leu 180 185 190 atg ggc atc ctg ctg cgg atc gcc agt ggg actcgc ctc tcc atc ccc 624 Met Gly Ile Leu Leu Arg Ile Ala Ser Gly Thr ArgLeu Ser Ile Pro 195 200 205 atc act tcc aac aca ttg atc tat tat cac ccccac ccc gaa gat att 672 Ile Thr Ser Asn Thr Leu Ile Tyr Tyr His Pro HisPro Glu Asp Ile 210 215 220 aag ttc cac ttg tac ctt gtc ccc agc gac gccttg cta aca aag gcg 720 Lys Phe His Leu Tyr Leu Val Pro Ser Asp Ala LeuLeu Thr Lys Ala 225 230 235 240 ata gat gat gag gaa gat cgc ttc cat ggtgtg cgc ctg cag act tcg 768 Ile Asp Asp Glu Glu Asp Arg Phe His Gly ValArg Leu Gln Thr Ser 245 250 255 ccc cca atg gaa ccc ctg aac ttt ggt tccagt tat att gtg tct aat 816 Pro Pro Met Glu Pro Leu Asn Phe Gly Ser SerTyr Ile Val Ser Asn 260 265 270 tct gct aac ctg aaa gta atg ccc aag gagttg aaa ttg tcc tac agg 864 Ser Ala Asn Leu Lys Val Met Pro Lys Glu LeuLys Leu Ser Tyr Arg 275 280 285 agc cct gga gaa att cag cac ttc tca aaattc tat gct ggg cag atg 912 Ser Pro Gly Glu Ile Gln His Phe Ser Lys PheTyr Ala Gly Gln Met 290 295 300 aag gaa ccc att caa ctt gag att act gaaaaa aga cat ggg act ttg 960 Lys Glu Pro Ile Gln Leu Glu Ile Thr Glu LysArg His Gly Thr Leu 305 310 315 320 gtg tgg gat act gag gtg aag cca gtggat ctc cag ctt gta gct gca 1008 Val Trp Asp Thr Glu Val Lys Pro Val AspLeu Gln Leu Val Ala Ala 325 330 335 tca gcc cct cct cct ttc tca ggt gcagcc ttt gtg aag gag aac cac 1056 Ser Ala Pro Pro Pro Phe Ser Gly Ala AlaPhe Val Lys Glu Asn His 340 345 350 cgg caa ctc caa gcc agg atg ggg gacctg aaa ggg gtg ctc gat gat 1104 Arg Gln Leu Gln Ala Arg Met Gly Asp LeuLys Gly Val Leu Asp Asp 355 360 365 ctc cag gac aat gag gtt ctt act gagaat gag aag gag ctg gtg gag 1152 Leu Gln Asp Asn Glu Val Leu Thr Glu AsnGlu Lys Glu Leu Val Glu 370 375 380 cag gaa aag aca cgg cag agc aag aatgag gcc ttg ctg agc atg gtg 1200 Gln Glu Lys Thr Arg Gln Ser Lys Asn GluAla Leu Leu Ser Met Val 385 390 395 400 gag aag aaa ggg gac ctg gcc ctggac gtg ctc ttc aga agc att agt 1248 Glu Lys Lys Gly Asp Leu Ala Leu AspVal Leu Phe Arg Ser Ile Ser 405 410 415 gaa agg gac cct tac ctc gtg tcctat ctt aga cag cag aat ttg taa 1296 Glu Arg Asp Pro Tyr Leu Val Ser TyrLeu Arg Gln Gln Asn Leu 420 425 430 aatgagtcag ttaggtagtc tggaagagagaatccagcgt tctcattgga aatggataaa 1356 cagaaatgtg atcattgatt tcagtgttcaagacagaaga agactgggta acatctatca 1416 cacaggcttt caggacagac ttgtaacctggcatgtacct attgactgta tcctcatgca 1476 ttttcctcaa g 1487 8 431 PRT Homosapiens 8 Met Met Arg Gln Arg Gln Ser His Tyr Cys Ser Val Leu Phe LeuSer 1 5 10 15 Val Asn Tyr Leu Gly Gly Thr Phe Pro Gly Asp Ile Cys SerGlu Glu 20 25 30 Asn Gln Ile Val Ser Ser Tyr Ala Ser Lys Val Cys Phe GluIle Glu 35 40 45 Glu Asp Tyr Lys Asn Arg Gln Phe Leu Gly Pro Glu Gly AsnVal Asp 50 55 60 Val Glu Leu Ile Asp Lys Ser Thr Asn Arg Tyr Ser Val TrpPhe Pro 65 70 75 80 Thr Ala Gly Trp Tyr Leu Trp Ser Ala Thr Gly Leu GlyPhe Leu Val 85 90 95 Arg Asp Glu Val Thr Val Thr Ile Ala Phe Gly Ser TrpSer Gln His 100 105 110 Leu Ala Leu Asp Leu Gln His His Glu Gln Trp LeuVal Gly Gly Pro 115 120 125 Leu Phe Asp Val Thr Ala Glu Pro Glu Glu AlaVal Ala Glu Ile His 130 135 140 Leu Pro His Phe Ile Ser Leu Gln Gly GluVal Asp Val Ser Trp Phe 145 150 155 160 Leu Val Ala His Phe Lys Asn GluGly Met Val Leu Glu His Pro Ala 165 170 175 Arg Val Glu Pro Phe Tyr AlaVal Leu Glu Ser Pro Ser Phe Ser Leu 180 185 190 Met Gly Ile Leu Leu ArgIle Ala Ser Gly Thr Arg Leu Ser Ile Pro 195 200 205 Ile Thr Ser Asn ThrLeu Ile Tyr Tyr His Pro His Pro Glu Asp Ile 210 215 220 Lys Phe His LeuTyr Leu Val Pro Ser Asp Ala Leu Leu Thr Lys Ala 225 230 235 240 Ile AspAsp Glu Glu Asp Arg Phe His Gly Val Arg Leu Gln Thr Ser 245 250 255 ProPro Met Glu Pro Leu Asn Phe Gly Ser Ser Tyr Ile Val Ser Asn 260 265 270Ser Ala Asn Leu Lys Val Met Pro Lys Glu Leu Lys Leu Ser Tyr Arg 275 280285 Ser Pro Gly Glu Ile Gln His Phe Ser Lys Phe Tyr Ala Gly Gln Met 290295 300 Lys Glu Pro Ile Gln Leu Glu Ile Thr Glu Lys Arg His Gly Thr Leu305 310 315 320 Val Trp Asp Thr Glu Val Lys Pro Val Asp Leu Gln Leu ValAla Ala 325 330 335 Ser Ala Pro Pro Pro Phe Ser Gly Ala Ala Phe Val LysGlu Asn His 340 345 350 Arg Gln Leu Gln Ala Arg Met Gly Asp Leu Lys GlyVal Leu Asp Asp 355 360 365 Leu Gln Asp Asn Glu Val Leu Thr Glu Asn GluLys Glu Leu Val Glu 370 375 380 Gln Glu Lys Thr Arg Gln Ser Lys Asn GluAla Leu Leu Ser Met Val 385 390 395 400 Glu Lys Lys Gly Asp Leu Ala LeuAsp Val Leu Phe Arg Ser Ile Ser 405 410 415 Glu Arg Asp Pro Tyr Leu ValSer Tyr Leu Arg Gln Gln Asn Leu 420 425 430 9 4556 DNA ArtificialSequence CDS (1)..(4362) Description of Artificial Sequence SyntheticConstruct 9 atg gct ggc gga gcc tgg ggc cgc ctg gcc tgt tac ttg gag ttcctg 48 Met Ala Gly Gly Ala Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 15 10 15 aag aag gag gag ctg aag gag ttc cag ctt ctg ctc gcc aat aaa gcg96 Lys Lys Glu Glu Leu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 2530 cac tcc agg agc tct tcg ggt gag aca ccc gct cag cca gag aag acg 144His Ser Arg Ser Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45agt ggc atg gag gtg gcc tcg tac ctg gtg gct cag tat ggg gag cag 192 SerGly Met Glu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 cgggcc tgg gac cta gcc ctc cat acc tgg gag cag atg ggg ctg agg 240 Arg AlaTrp Asp Leu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80 tcactg tgc gcc caa gcc cag gaa ggg gca ggc cac tct ccc tca ttc 288 Ser LeuCys Ala Gln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 90 95 ccc tacagc cca agt gaa ccc cac ctg ggg tct ccc agc caa ccc acc 336 Pro Tyr SerPro Ser Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100 105 110 tcc accgca gtg cta atg ccc tgg atc cat gaa ttg ccg gcg ggg tgc 384 Ser Thr AlaVal Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115 120 125 acc cagggc tca gag aga agg gtt ttg aga cag ctg cct gac aca tct 432 Thr Gln GlySer Glu Arg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser 130 135 140 gga cgccgc tgg aga gaa atc tct gcc tca ctc ctc tac caa gct ctt 480 Gly Arg ArgTrp Arg Glu Ile Ser Ala Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 ccaagc tcc cca gac cat gag tct cca agc cag gag tca ccc aac gcc 528 Pro SerSer Pro Asp His Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 cccaca tcc aca gca gtg ctg ggg agc tgg gga tcc cca cct cag ccc 576 Pro ThrSer Thr Ala Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 agccta gca ccc aga gag cag gag gct cct ggg acc caa tgg cct ctg 624 Ser LeuAla Pro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205 gatgaa acg tca gga att tac tac aca gaa atc aga gaa aga gag aga 672 Asp GluThr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215 220 gagaaa tca gag aaa ggc agg ccc cca tgg gca gcg gtg gta gga acg 720 Glu LysSer Glu Lys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr 225 230 235 240ccc cca cag gcg cac acc agc cta cag ccc cac cac cac cca tgg gag 768 ProPro Gln Ala His Thr Ser Leu Gln Pro His His His Pro Trp Glu 245 250 255cct tct gtg aga gag agc ctc tgt tcc aca tgg ccc tgg aaa aat gag 816 ProSer Val Arg Glu Ser Leu Cys Ser Thr Trp Pro Trp Lys Asn Glu 260 265 270gat ttt aac caa aaa ttc aca cag ctg cta ctt cta caa aga cct cac 864 AspPhe Asn Gln Lys Phe Thr Gln Leu Leu Leu Leu Gln Arg Pro His 275 280 285ccc aga agc caa gat ccc ctg gtc aag aga agc tgg cct gat tat gtg 912 ProArg Ser Gln Asp Pro Leu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300gag gag aat cga gga cat tta att gag atc aga gac tta ttt ggc cca 960 GluGlu Asn Arg Gly His Leu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315320 ggc ctg gat acc caa gaa cct cgc ata gtc ata ctg cag ggg gct gct 1008Gly Leu Asp Thr Gln Glu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330335 gga att ggg aag tca aca ctg gcc agg cag gtg aag gaa gcc tgg ggg 1056Gly Ile Gly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345350 aga ggc cag ctg tat ggg gac cgc ttc cag cat gtc ttc tac ttc agc 1104Arg Gly Gln Leu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355 360365 tgc aga gag ctg gcc cag tcc aag gtg gtg agt ctc gct gag ctc atc 1152Cys Arg Glu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu Leu Ile 370 375380 gga aaa gat ggg aca gcc act ccg gct ccc att aga cag atc ctg tct 1200Gly Lys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385 390395 400 agg cca gag cgg ctg ctc ttc atc ctc gat ggt gta gat gag cca gga1248 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405410 415 tgg gtc ttg cag gag ccg agt tct gag ctc tgt ctg cac tgg agc cag1296 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420425 430 cca cag ccg gcg gat gca ctg ctg ggc agt ttg ctg ggg aaa act ata1344 Pro Gln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435440 445 ctt ccc gag gca tcc ttc ctg atc acg gct cgg acc aca gct ctg cag1392 Leu Pro Glu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450455 460 aac ctc att cct tct ttg gag cag gca cgt tgg gta gag gtc ctg ggg1440 Asn Leu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465470 475 480 ttc tct gag tcc agc agg aag gaa tat ttc tac aga tat ttc acagat 1488 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp485 490 495 gaa agg caa gca att aga gcc ttt agg ttg gtc aaa tca aac aaagag 1536 Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu500 505 510 ctc tgg gcc ctg tgt ctt gtg ccc tgg gtg tcc tgg ctg gcc tgcact 1584 Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser Trp Leu Ala Cys Thr515 520 525 tgc ctg atg cag cag atg aag cgg aag gaa aaa ctc aca ctg acttcc 1632 Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys Leu Thr Leu Thr Ser530 535 540 aag acc acc aca acc ctc tgt cta cat tac ctt gcc cag gct ctccaa 1680 Lys Thr Thr Thr Thr Leu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln545 550 555 560 gct cag cca ttg gga ccc cag ctc aga gac ctc tgc tct ctggct gct 1728 Ala Gln Pro Leu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu AlaAla 565 570 575 gag ggc atc tgg caa aaa aag acc ctt ttc agt cca gat gacctc agg 1776 Glu Gly Ile Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp LeuArg 580 585 590 aag cat ggg tta gat ggg gcc atc atc tcc acc ttc ttg aagatg ggt 1824 Lys His Gly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys MetGly 595 600 605 att ctt caa gag cac ccc atc cct ctg agc tac agc ttc attcac ctc 1872 Ile Leu Gln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile HisLeu 610 615 620 tgt ttc caa gag ttc ttt gca gca atg tcc tat gtc ttg gaggat gag 1920 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu AspGlu 625 630 635 640 aag ggg aga ggt aaa cat tct aat tgc atc ata gat ttggaa aag acg 1968 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile Asp Leu GluLys Thr 645 650 655 cta gaa gca tat gga ata cat ggc ctg ttt ggg gca tcaacc aca cgt 2016 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe Gly Ala Ser ThrThr Arg 660 665 670 ttc cta ttg ggc ctg tta agt gat gag ggg gag aga gagatg gag aac 2064 Phe Leu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg Glu MetGlu Asn 675 680 685 atc ttt cac tgc cgg ctg tct cag ggg agg aac ctg atgcag tgg gtc 2112 Ile Phe His Cys Arg Leu Ser Gln Gly Arg Asn Leu Met GlnTrp Val 690 695 700 ccg tcc ctg cag ctg ctg ctg cag cca cac tct ctg gagtcc ctc cac 2160 Pro Ser Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu SerLeu His 705 710 715 720 tgc ttg tac gag act cgg aac aaa acg ttc ctg acacaa gtg atg gcc 2208 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr GlnVal Met Ala 725 730 735 cat ttc gaa gaa atg ggc atg tgt gta gaa aca gacatg gag ctc tta 2256 His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp MetGlu Leu Leu 740 745 750 gtg tgc act ttc tgc att aaa ttc agc cgc cac gtgaag aag ctt cag 2304 Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val LysLys Leu Gln 755 760 765 ctg att gag ggc agg cag cac aga tca aca tgg agcccc acc atg gta 2352 Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp Ser ProThr Met Val 770 775 780 gtc ctg ttc agg tgg gtc cca gtc aca gat gcc tattgg cag att ctc 2400 Val Leu Phe Arg Trp Val Pro Val Thr Asp Ala Tyr TrpGln Ile Leu 785 790 795 800 ttc tcc gtc ctc aag gtc acc aga aac ctg aaggag ctg gac cta agt 2448 Phe Ser Val Leu Lys Val Thr Arg Asn Leu Lys GluLeu Asp Leu Ser 805 810 815 gga aac tcg ctg agc cac tct gca gtg aag agtctt tgt aag acc ctg 2496 Gly Asn Ser Leu Ser His Ser Ala Val Lys Ser LeuCys Lys Thr Leu 820 825 830 aga cgc cct cgc tgc ctc ctg gag acc ctg cggttg gct ggc tgt ggc 2544 Arg Arg Pro Arg Cys Leu Leu Glu Thr Leu Arg LeuAla Gly Cys Gly 835 840 845 ctc aca gct gag gac tgc aag gac ctt gcc tttggg ctg aga gcc aac 2592 Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe GlyLeu Arg Ala Asn 850 855 860 cag acc ctg acc gag ctg gac ctg agc ttc aatgtg ctc acg gat gct 2640 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn ValLeu Thr Asp Ala 865 870 875 880 gga gcc aaa cac ctt tgc cag aga ctg agacag ccg agc tgc aag cta 2688 Gly Ala Lys His Leu Cys Gln Arg Leu Arg GlnPro Ser Cys Lys Leu 885 890 895 cag cga ctg cag ctg gtc agc tgt ggc ctcacg tct gac tgc tgc cag 2736 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu ThrSer Asp Cys Cys Gln 900 905 910 gac ctg gcc tct gtg ctt agt gcc agc cccagc ctg aag gag cta gac 2784 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro SerLeu Lys Glu Leu Asp 915 920 925 ctg cag cag aac aac ctg gat gac gtt ggcgtg cga ctg ctc tgt gag 2832 Leu Gln Gln Asn Asn Leu Asp Asp Val Gly ValArg Leu Leu Cys Glu 930 935 940 ggg ctc agg cat cct gcc tgc aaa ctc atacgc ctg ggg ctg gac cag 2880 Gly Leu Arg His Pro Ala Cys Lys Leu Ile ArgLeu Gly Leu Asp Gln 945 950 955 960 aca act ctg agt gat gag atg agg caggaa ctg agg gcc ctg gag cag 2928 Thr Thr Leu Ser Asp Glu Met Arg Gln GluLeu Arg Ala Leu Glu Gln 965 970 975 gag aaa cct cag ctg ctc atc ttc agcaga cgg aaa cca agt gtg atg 2976 Glu Lys Pro Gln Leu Leu Ile Phe Ser ArgArg Lys Pro Ser Val Met 980 985 990 acc cct act gag ggc ctg gat acg ggagag atg agt aat agc aca tcc 3024 Thr Pro Thr Glu Gly Leu Asp Thr Gly GluMet Ser Asn Ser Thr Ser 995 1000 1005 tca ctc aag cgg cag aga ctc ggatca gag agg gcg gct tcc cat gtt 3072 Ser Leu Lys Arg Gln Arg Leu Gly SerGlu Arg Ala Ala Ser His Val 1010 1015 1020 gct cag gct aat ctc aaa ctcctg gac gtg agc aag atc ttc cca att 3120 Ala Gln Ala Asn Leu Lys Leu LeuAsp Val Ser Lys Ile Phe Pro Ile 1025 1030 1035 1040 gct gag att gca gaggaa agc tcc cca gag gta gta ccg gtg gaa ctc 3168 Ala Glu Ile Ala Glu GluSer Ser Pro Glu Val Val Pro Val Glu Leu 1045 1050 1055 ttg tgc gtg ccttct cct gcc tct caa ggg gac ctg cat acg aag cct 3216 Leu Cys Val Pro SerPro Ala Ser Gln Gly Asp Leu His Thr Lys Pro 1060 1065 1070 ttg ggg actgac gat gac ttt ctg ggg cct gaa gga aat gtg gat gtt 3264 Leu Gly Thr AspAsp Asp Phe Leu Gly Pro Glu Gly Asn Val Asp Val 1075 1080 1085 gag ttgatt gat aag agc aca aac aga tac agc gtt tgg ttc ccc act 3312 Glu Leu IleAsp Lys Ser Thr Asn Arg Tyr Ser Val Trp Phe Pro Thr 1090 1095 1100 gctggc tgg tat ctg tgg tca gcc aca ggc ctc ggc ttc ctg gta agg 3360 Ala GlyTrp Tyr Leu Trp Ser Ala Thr Gly Leu Gly Phe Leu Val Arg 1105 1110 11151120 gat gag gtc aca gtg acg att gcg ttt ggt tcc tgg agt cag cac ctg3408 Asp Glu Val Thr Val Thr Ile Ala Phe Gly Ser Trp Ser Gln His Leu1125 1130 1135 gcc ctg gac ctg cag cac cat gaa cag tgg ctg gtg ggc ggcccc ttg 3456 Ala Leu Asp Leu Gln His His Glu Gln Trp Leu Val Gly Gly ProLeu 1140 1145 1150 ttt gat gtc act gca gag cca gag gag gct gtc gcc gaaatc cac ctc 3504 Phe Asp Val Thr Ala Glu Pro Glu Glu Ala Val Ala Glu IleHis Leu 1155 1160 1165 ccc cac ttc atc tcc ctc caa ggt gag gtg gac gtctcc tgg ttt ctc 3552 Pro His Phe Ile Ser Leu Gln Gly Glu Val Asp Val SerTrp Phe Leu 1170 1175 1180 gtt gcc cat ttt aag aat gaa ggg atg gtc ctggag cat cca gcc cgg 3600 Val Ala His Phe Lys Asn Glu Gly Met Val Leu GluHis Pro Ala Arg 1185 1190 1195 1200 gtg gag cct ttc tat gct gtc ctg gaaagc ccc agc ttc tct ctg atg 3648 Val Glu Pro Phe Tyr Ala Val Leu Glu SerPro Ser Phe Ser Leu Met 1205 1210 1215 ggc atc ctg ctg cgg atc gcc agtggg act cgc ctc tcc atc ccc atc 3696 Gly Ile Leu Leu Arg Ile Ala Ser GlyThr Arg Leu Ser Ile Pro Ile 1220 1225 1230 act tcc aac aca ttg atc tattat cac ccc cac ccc gaa gat att aag 3744 Thr Ser Asn Thr Leu Ile Tyr TyrHis Pro His Pro Glu Asp Ile Lys 1235 1240 1245 ttc cac ttg tac ctt gtcccc agc gac gcc ttg cta aca aag gcg ata 3792 Phe His Leu Tyr Leu Val ProSer Asp Ala Leu Leu Thr Lys Ala Ile 1250 1255 1260 gat gat gag gaa gatcgc ttc cat ggt gtg cgc ctg cag act tcg ccc 3840 Asp Asp Glu Glu Asp ArgPhe His Gly Val Arg Leu Gln Thr Ser Pro 1265 1270 1275 1280 cca atg gaaccc ctg aac ttt ggt tcc agt tat att gtg tct aat tct 3888 Pro Met Glu ProLeu Asn Phe Gly Ser Ser Tyr Ile Val Ser Asn Ser 1285 1290 1295 gct aacctg aaa gta atg ccc aag gag ttg aaa ttg tcc tac agg agc 3936 Ala Asn LeuLys Val Met Pro Lys Glu Leu Lys Leu Ser Tyr Arg Ser 1300 1305 1310 cctgga gaa att cag cac ttc tca aaa ttc tat gct ggg cag atg aag 3984 Pro GlyGlu Ile Gln His Phe Ser Lys Phe Tyr Ala Gly Gln Met Lys 1315 1320 1325gaa ccc att caa ctt gag att act gaa aaa aga cat ggg act ttg gtg 4032 GluPro Ile Gln Leu Glu Ile Thr Glu Lys Arg His Gly Thr Leu Val 1330 13351340 tgg gat act gag gtg aag cca gtg gat ctc cag ctt gta gct gca tca4080 Trp Asp Thr Glu Val Lys Pro Val Asp Leu Gln Leu Val Ala Ala Ser1345 1350 1355 1360 gcc cct cct cct ttc tca ggt gca gcc ttt gtg aag gagaac cac cgg 4128 Ala Pro Pro Pro Phe Ser Gly Ala Ala Phe Val Lys Glu AsnHis Arg 1365 1370 1375 caa ctc caa gcc agg atg ggg gac ctg aaa ggg gtgctc gat gat ctc 4176 Gln Leu Gln Ala Arg Met Gly Asp Leu Lys Gly Val LeuAsp Asp Leu 1380 1385 1390 cag gac aat gag gtt ctt act gag aat gag aaggag ctg gtg gag cag 4224 Gln Asp Asn Glu Val Leu Thr Glu Asn Glu Lys GluLeu Val Glu Gln 1395 1400 1405 gaa aag aca cgg cag agc aag aat gag gccttg ctg agc atg gtg gag 4272 Glu Lys Thr Arg Gln Ser Lys Asn Glu Ala LeuLeu Ser Met Val Glu 1410 1415 1420 aag aaa ggg gac ctg gcc ctg gac gtgctc ttc aga agc att agt gaa 4320 Lys Lys Gly Asp Leu Ala Leu Asp Val LeuPhe Arg Ser Ile Ser Glu 1425 1430 1435 1440 agg gac cct tac ctc gtg tcctat ctt aga cag cag aat ttg 4362 Arg Asp Pro Tyr Leu Val Ser Tyr Leu ArgGln Gln Asn Leu 1445 1450 taaaatgagt cagttaggta gtctggaaga gagaatccagcgttctcatt ggaaatggat 4422 aaacagaaat gtgatcattg atttcagtgt tcaagacagaagaagactgg gtaacatcta 4482 tcacacaggc tttcaggaca gacttgtaac ctggcatgtacctattgact gtatcctcat 4542 gcattttcct caag 4556 10 1454 PRT ArtificialSequence Description of Artificial Sequence Synthetic 10 Met Ala Gly GlyAla Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15 Lys Lys GluGlu Leu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 His Ser ArgSer Ser Ser Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45 Ser Gly MetGlu Val Ala Ser Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 Arg Ala TrpAsp Leu Ala Leu His Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80 Ser LeuCys Ala Gln Ala Gln Glu Gly Ala Gly His Ser Pro Ser Phe 85 90 95 Pro TyrSer Pro Ser Glu Pro His Leu Gly Ser Pro Ser Gln Pro Thr 100 105 110 SerThr Ala Val Leu Met Pro Trp Ile His Glu Leu Pro Ala Gly Cys 115 120 125Thr Gln Gly Ser Glu Arg Arg Val Leu Arg Gln Leu Pro Asp Thr Ser 130 135140 Gly Arg Arg Trp Arg Glu Ile Ser Ala Ser Leu Leu Tyr Gln Ala Leu 145150 155 160 Pro Ser Ser Pro Asp His Glu Ser Pro Ser Gln Glu Ser Pro AsnAla 165 170 175 Pro Thr Ser Thr Ala Val Leu Gly Ser Trp Gly Ser Pro ProGln Pro 180 185 190 Ser Leu Ala Pro Arg Glu Gln Glu Ala Pro Gly Thr GlnTrp Pro Leu 195 200 205 Asp Glu Thr Ser Gly Ile Tyr Tyr Thr Glu Ile ArgGlu Arg Glu Arg 210 215 220 Glu Lys Ser Glu Lys Gly Arg Pro Pro Trp AlaAla Val Val Gly Thr 225 230 235 240 Pro Pro Gln Ala His Thr Ser Leu GlnPro His His His Pro Trp Glu 245 250 255 Pro Ser Val Arg Glu Ser Leu CysSer Thr Trp Pro Trp Lys Asn Glu 260 265 270 Asp Phe Asn Gln Lys Phe ThrGln Leu Leu Leu Leu Gln Arg Pro His 275 280 285 Pro Arg Ser Gln Asp ProLeu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300 Glu Glu Asn Arg GlyHis Leu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320 Gly Leu AspThr Gln Glu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330 335 Gly IleGly Lys Ser Thr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345 350 ArgGly Gln Leu Tyr Gly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355 360 365Cys Arg Glu Leu Ala Gln Ser Lys Val Val Ser Leu Ala Glu Leu Ile 370 375380 Gly Lys Asp Gly Thr Ala Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385390 395 400 Arg Pro Glu Arg Leu Leu Phe Ile Leu Asp Gly Val Asp Glu ProGly 405 410 415 Trp Val Leu Gln Glu Pro Ser Ser Glu Leu Cys Leu His TrpSer Gln 420 425 430 Pro Gln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu GlyLys Thr Ile 435 440 445 Leu Pro Glu Ala Ser Phe Leu Ile Thr Ala Arg ThrThr Ala Leu Gln 450 455 460 Asn Leu Ile Pro Ser Leu Glu Gln Ala Arg TrpVal Glu Val Leu Gly 465 470 475 480 Phe Ser Glu Ser Ser Arg Lys Glu TyrPhe Tyr Arg Tyr Phe Thr Asp 485 490 495 Glu Arg Gln Ala Ile Arg Ala PheArg Leu Val Lys Ser Asn Lys Glu 500 505 510 Leu Trp Ala Leu Cys Leu ValPro Trp Val Ser Trp Leu Ala Cys Thr 515 520 525 Cys Leu Met Gln Gln MetLys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535 540 Lys Thr Thr Thr ThrLeu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560 Ala Gln ProLeu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570 575 Glu GlyIle Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580 585 590 LysHis Gly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595 600 605Ile Leu Gln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu 610 615620 Cys Phe Gln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625630 635 640 Lys Gly Arg Gly Lys His Ser Asn Cys Ile Ile Asp Leu Glu LysThr 645 650 655 Leu Glu Ala Tyr Gly Ile His Gly Leu Phe Gly Ala Ser ThrThr Arg 660 665 670 Phe Leu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg GluMet Glu Asn 675 680 685 Ile Phe His Cys Arg Leu Ser Gln Gly Arg Asn LeuMet Gln Trp Val 690 695 700 Pro Ser Leu Gln Leu Leu Leu Gln Pro His SerLeu Glu Ser Leu His 705 710 715 720 Cys Leu Tyr Glu Thr Arg Asn Lys ThrPhe Leu Thr Gln Val Met Ala 725 730 735 His Phe Glu Glu Met Gly Met CysVal Glu Thr Asp Met Glu Leu Leu 740 745 750 Val Cys Thr Phe Cys Ile LysPhe Ser Arg His Val Lys Lys Leu Gln 755 760 765 Leu Ile Glu Gly Arg GlnHis Arg Ser Thr Trp Ser Pro Thr Met Val 770 775 780 Val Leu Phe Arg TrpVal Pro Val Thr Asp Ala Tyr Trp Gln Ile Leu 785 790 795 800 Phe Ser ValLeu Lys Val Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805 810 815 Gly AsnSer Leu Ser His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820 825 830 ArgArg Pro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly 835 840 845Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg Ala Asn 850 855860 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu Thr Asp Ala 865870 875 880 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln Pro Ser Cys LysLeu 885 890 895 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu Thr Ser Asp CysCys Gln 900 905 910 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu LysGlu Leu Asp 915 920 925 Leu Gln Gln Asn Asn Leu Asp Asp Val Gly Val ArgLeu Leu Cys Glu 930 935 940 Gly Leu Arg His Pro Ala Cys Lys Leu Ile ArgLeu Gly Leu Asp Gln 945 950 955 960 Thr Thr Leu Ser Asp Glu Met Arg GlnGlu Leu Arg Ala Leu Glu Gln 965 970 975 Glu Lys Pro Gln Leu Leu Ile PheSer Arg Arg Lys Pro Ser Val Met 980 985 990 Thr Pro Thr Glu Gly Leu AspThr Gly Glu Met Ser Asn Ser Thr Ser 995 1000 1005 Ser Leu Lys Arg GlnArg Leu Gly Ser Glu Arg Ala Ala Ser His Val 1010 1015 1020 Ala Gln AlaAsn Leu Lys Leu Leu Asp Val Ser Lys Ile Phe Pro Ile 1025 1030 1035 1040Ala Glu Ile Ala Glu Glu Ser Ser Pro Glu Val Val Pro Val Glu Leu 10451050 1055 Leu Cys Val Pro Ser Pro Ala Ser Gln Gly Asp Leu His Thr LysPro 1060 1065 1070 Leu Gly Thr Asp Asp Asp Phe Leu Gly Pro Glu Gly AsnVal Asp Val 1075 1080 1085 Glu Leu Ile Asp Lys Ser Thr Asn Arg Tyr SerVal Trp Phe Pro Thr 1090 1095 1100 Ala Gly Trp Tyr Leu Trp Ser Ala ThrGly Leu Gly Phe Leu Val Arg 1105 1110 1115 1120 Asp Glu Val Thr Val ThrIle Ala Phe Gly Ser Trp Ser Gln His Leu 1125 1130 1135 Ala Leu Asp LeuGln His His Glu Gln Trp Leu Val Gly Gly Pro Leu 1140 1145 1150 Phe AspVal Thr Ala Glu Pro Glu Glu Ala Val Ala Glu Ile His Leu 1155 1160 1165Pro His Phe Ile Ser Leu Gln Gly Glu Val Asp Val Ser Trp Phe Leu 11701175 1180 Val Ala His Phe Lys Asn Glu Gly Met Val Leu Glu His Pro AlaArg 1185 1190 1195 1200 Val Glu Pro Phe Tyr Ala Val Leu Glu Ser Pro SerPhe Ser Leu Met 1205 1210 1215 Gly Ile Leu Leu Arg Ile Ala Ser Gly ThrArg Leu Ser Ile Pro Ile 1220 1225 1230 Thr Ser Asn Thr Leu Ile Tyr TyrHis Pro His Pro Glu Asp Ile Lys 1235 1240 1245 Phe His Leu Tyr Leu ValPro Ser Asp Ala Leu Leu Thr Lys Ala Ile 1250 1255 1260 Asp Asp Glu GluAsp Arg Phe His Gly Val Arg Leu Gln Thr Ser Pro 1265 1270 1275 1280 ProMet Glu Pro Leu Asn Phe Gly Ser Ser Tyr Ile Val Ser Asn Ser 1285 12901295 Ala Asn Leu Lys Val Met Pro Lys Glu Leu Lys Leu Ser Tyr Arg Ser1300 1305 1310 Pro Gly Glu Ile Gln His Phe Ser Lys Phe Tyr Ala Gly GlnMet Lys 1315 1320 1325 Glu Pro Ile Gln Leu Glu Ile Thr Glu Lys Arg HisGly Thr Leu Val 1330 1335 1340 Trp Asp Thr Glu Val Lys Pro Val Asp LeuGln Leu Val Ala Ala Ser 1345 1350 1355 1360 Ala Pro Pro Pro Phe Ser GlyAla Ala Phe Val Lys Glu Asn His Arg 1365 1370 1375 Gln Leu Gln Ala ArgMet Gly Asp Leu Lys Gly Val Leu Asp Asp Leu 1380 1385 1390 Gln Asp AsnGlu Val Leu Thr Glu Asn Glu Lys Glu Leu Val Glu Gln 1395 1400 1405 GluLys Thr Arg Gln Ser Lys Asn Glu Ala Leu Leu Ser Met Val Glu 1410 14151420 Lys Lys Gly Asp Leu Ala Leu Asp Val Leu Phe Arg Ser Ile Ser Glu1425 1430 1435 1440 Arg Asp Pro Tyr Leu Val Ser Tyr Leu Arg Gln Gln AsnLeu 1445 1450 11 4466 DNA Artificial Sequence CDS (1)..(4272)Description of Artificial Sequence Synthetic Construct 11 atg gct ggcgga gcc tgg ggc cgc ctg gcc tgt tac ttg gag ttc ctg 48 Met Ala Gly GlyAla Trp Gly Arg Leu Ala Cys Tyr Leu Glu Phe Leu 1 5 10 15 aag aag gaggag ctg aag gag ttc cag ctt ctg ctc gcc aat aaa gcg 96 Lys Lys Glu GluLeu Lys Glu Phe Gln Leu Leu Leu Ala Asn Lys Ala 20 25 30 cac tcc agg agctct tcg ggt gag aca ccc gct cag cca gag aag acg 144 His Ser Arg Ser SerSer Gly Glu Thr Pro Ala Gln Pro Glu Lys Thr 35 40 45 agt ggc atg gag gtggcc tcg tac ctg gtg gct cag tat ggg gag cag 192 Ser Gly Met Glu Val AlaSer Tyr Leu Val Ala Gln Tyr Gly Glu Gln 50 55 60 cgg gcc tgg gac cta gccctc cat acc tgg gag cag atg ggg ctg agg 240 Arg Ala Trp Asp Leu Ala LeuHis Thr Trp Glu Gln Met Gly Leu Arg 65 70 75 80 tca ctg tgc gcc caa gcccag gaa ggg gca ggc cac tct ccc tca ttc 288 Ser Leu Cys Ala Gln Ala GlnGlu Gly Ala Gly His Ser Pro Ser Phe 85 90 95 ccc tac agc cca agt gaa ccccac ctg ggg tct ccc agc caa ccc acc 336 Pro Tyr Ser Pro Ser Glu Pro HisLeu Gly Ser Pro Ser Gln Pro Thr 100 105 110 tcc acc gca gtg cta atg ccctgg atc cat gaa ttg ccg gcg ggg tgc 384 Ser Thr Ala Val Leu Met Pro TrpIle His Glu Leu Pro Ala Gly Cys 115 120 125 acc cag ggc tca gag aga agggtt ttg aga cag ctg cct gac aca tct 432 Thr Gln Gly Ser Glu Arg Arg ValLeu Arg Gln Leu Pro Asp Thr Ser 130 135 140 gga cgc cgc tgg aga gaa atctct gcc tca ctc ctc tac caa gct ctt 480 Gly Arg Arg Trp Arg Glu Ile SerAla Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 cca agc tcc cca gac catgag tct cca agc cag gag tca ccc aac gcc 528 Pro Ser Ser Pro Asp His GluSer Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 ccc aca tcc aca gca gtgctg ggg agc tgg gga tcc cca cct cag ccc 576 Pro Thr Ser Thr Ala Val LeuGly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 agc cta gca ccc aga gagcag gag gct cct ggg acc caa tgg cct ctg 624 Ser Leu Ala Pro Arg Glu GlnGlu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205 gat gaa acg tca gga atttac tac aca gaa atc aga gaa aga gag aga 672 Asp Glu Thr Ser Gly Ile TyrTyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215 220 gag aaa tca gag aaa ggcagg ccc cca tgg gca gcg gtg gta gga acg 720 Glu Lys Ser Glu Lys Gly ArgPro Pro Trp Ala Ala Val Val Gly Thr 225 230 235 240 ccc cca cag gcg cacacc agc cta cag ccc cac cac cac cca tgg gag 768 Pro Pro Gln Ala His ThrSer Leu Gln Pro His His His Pro Trp Glu 245 250 255 cct tct gtg aga gagagc ctc tgt tcc aca tgg ccc tgg aaa aat gag 816 Pro Ser Val Arg Glu SerLeu Cys Ser Thr Trp Pro Trp Lys Asn Glu 260 265 270 gat ttt aac caa aaattc aca cag ctg cta ctt cta caa aga cct cac 864 Asp Phe Asn Gln Lys PheThr Gln Leu Leu Leu Leu Gln Arg Pro His 275 280 285 ccc aga agc caa gatccc ctg gtc aag aga agc tgg cct gat tat gtg 912 Pro Arg Ser Gln Asp ProLeu Val Lys Arg Ser Trp Pro Asp Tyr Val 290 295 300 gag gag aat cga ggacat tta att gag atc aga gac tta ttt ggc cca 960 Glu Glu Asn Arg Gly HisLeu Ile Glu Ile Arg Asp Leu Phe Gly Pro 305 310 315 320 ggc ctg gat acccaa gaa cct cgc ata gtc ata ctg cag ggg gct gct 1008 Gly Leu Asp Thr GlnGlu Pro Arg Ile Val Ile Leu Gln Gly Ala Ala 325 330 335 gga att ggg aagtca aca ctg gcc agg cag gtg aag gaa gcc tgg ggg 1056 Gly Ile Gly Lys SerThr Leu Ala Arg Gln Val Lys Glu Ala Trp Gly 340 345 350 aga ggc cag ctgtat ggg gac cgc ttc cag cat gtc ttc tac ttc agc 1104 Arg Gly Gln Leu TyrGly Asp Arg Phe Gln His Val Phe Tyr Phe Ser 355 360 365 tgc aga gag ctggcc cag tcc aag gtg gtg agt ctc gct gag ctc atc 1152 Cys Arg Glu Leu AlaGln Ser Lys Val Val Ser Leu Ala Glu Leu Ile 370 375 380 gga aaa gat gggaca gcc act ccg gct ccc att aga cag atc ctg tct 1200 Gly Lys Asp Gly ThrAla Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395 400 agg cca gagcgg ctg ctc ttc atc ctc gat ggt gta gat gag cca gga 1248 Arg Pro Glu ArgLeu Leu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405 410 415 tgg gtc ttgcag gag ccg agt tct gag ctc tgt ctg cac tgg agc cag 1296 Trp Val Leu GlnGlu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420 425 430 cca cag ccggcg gat gca ctg ctg ggc agt ttg ctg ggg aaa act ata 1344 Pro Gln Pro AlaAsp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445 ctt ccc gaggca tcc ttc ctg atc acg gct cgg acc aca gct ctg cag 1392 Leu Pro Glu AlaSer Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450 455 460 aac ctc attcct tct ttg gag cag gca cgt tgg gta gag gtc ctg ggg 1440 Asn Leu Ile ProSer Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465 470 475 480 ttc tctgag tcc agc agg aag gaa tat ttc tac aga tat ttc aca gat 1488 Phe Ser GluSer Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe Thr Asp 485 490 495 gaa aggcaa gca att aga gcc ttt agg ttg gtc aaa tca aac aaa gag 1536 Glu Arg GlnAla Ile Arg Ala Phe Arg Leu Val Lys Ser Asn Lys Glu 500 505 510 ctc tgggcc ctg tgt ctt gtg ccc tgg gtg tcc tgg ctg gcc tgc act 1584 Leu Trp AlaLeu Cys Leu Val Pro Trp Val Ser Trp Leu Ala Cys Thr 515 520 525 tgc ctgatg cag cag atg aag cgg aag gaa aaa ctc aca ctg act tcc 1632 Cys Leu MetGln Gln Met Lys Arg Lys Glu Lys Leu Thr Leu Thr Ser 530 535 540 aag accacc aca acc ctc tgt cta cat tac ctt gcc cag gct ctc caa 1680 Lys Thr ThrThr Thr Leu Cys Leu His Tyr Leu Ala Gln Ala Leu Gln 545 550 555 560 gctcag cca ttg gga ccc cag ctc aga gac ctc tgc tct ctg gct gct 1728 Ala GlnPro Leu Gly Pro Gln Leu Arg Asp Leu Cys Ser Leu Ala Ala 565 570 575 gagggc atc tgg caa aaa aag acc ctt ttc agt cca gat gac ctc agg 1776 Glu GlyIle Trp Gln Lys Lys Thr Leu Phe Ser Pro Asp Asp Leu Arg 580 585 590 aagcat ggg tta gat ggg gcc atc atc tcc acc ttc ttg aag atg ggt 1824 Lys HisGly Leu Asp Gly Ala Ile Ile Ser Thr Phe Leu Lys Met Gly 595 600 605 attctt caa gag cac ccc atc cct ctg agc tac agc ttc att cac ctc 1872 Ile LeuGln Glu His Pro Ile Pro Leu Ser Tyr Ser Phe Ile His Leu 610 615 620 tgtttc caa gag ttc ttt gca gca atg tcc tat gtc ttg gag gat gag 1920 Cys PheGln Glu Phe Phe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625 630 635 640aag ggg aga ggt aaa cat tct aat tgc atc ata gat ttg gaa aag acg 1968 LysGly Arg Gly Lys His Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645 650 655cta gaa gca tat gga ata cat ggc ctg ttt ggg gca tca acc aca cgt 2016 LeuGlu Ala Tyr Gly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg 660 665 670ttc cta ttg ggc ctg tta agt gat gag ggg gag aga gag atg gag aac 2064 PheLeu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn 675 680 685atc ttt cac tgc cgg ctg tct cag ggg agg aac ctg atg cag tgg gtc 2112 IlePhe His Cys Arg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val 690 695 700ccg tcc ctg cag ctg ctg ctg cag cca cac tct ctg gag tcc ctc cac 2160 ProSer Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His 705 710 715720 tgc ttg tac gag act cgg aac aaa acg ttc ctg aca caa gtg atg gcc 2208Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val Met Ala 725 730735 cat ttc gaa gaa atg ggc atg tgt gta gaa aca gac atg gag ctc tta 2256His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp Met Glu Leu Leu 740 745750 gtg tgc act ttc tgc att aaa ttc agc cgc cac gtg aag aag ctt cag 2304Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val Lys Lys Leu Gln 755 760765 ctg att gag ggc agg cag cac aga tca aca tgg agc ccc acc atg gta 2352Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp Ser Pro Thr Met Val 770 775780 gtc ctg ttc agg tgg gtc cca gtc aca gat gcc tat tgg cag att ctc 2400Val Leu Phe Arg Trp Val Pro Val Thr Asp Ala Tyr Trp Gln Ile Leu 785 790795 800 ttc tcc gtc ctc aag gtc acc aga aac ctg aag gag ctg gac cta agt2448 Phe Ser Val Leu Lys Val Thr Arg Asn Leu Lys Glu Leu Asp Leu Ser 805810 815 gga aac tcg ctg agc cac tct gca gtg aag agt ctt tgt aag acc ctg2496 Gly Asn Ser Leu Ser His Ser Ala Val Lys Ser Leu Cys Lys Thr Leu 820825 830 aga cgc cct cgc tgc ctc ctg gag acc ctg cgg ttg gct ggc tgt ggc2544 Arg Arg Pro Arg Cys Leu Leu Glu Thr Leu Arg Leu Ala Gly Cys Gly 835840 845 ctc aca gct gag gac tgc aag gac ctt gcc ttt ggg ctg aga gcc aac2592 Leu Thr Ala Glu Asp Cys Lys Asp Leu Ala Phe Gly Leu Arg Ala Asn 850855 860 cag acc ctg acc gag ctg gac ctg agc ttc aat gtg ctc acg gat gct2640 Gln Thr Leu Thr Glu Leu Asp Leu Ser Phe Asn Val Leu Thr Asp Ala 865870 875 880 gga gcc aaa cac ctt tgc cag aga ctg aga cag ccg agc tgc aagcta 2688 Gly Ala Lys His Leu Cys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu885 890 895 cag cga ctg cag ctg gtc agc tgt ggc ctc acg tct gac tgc tgccag 2736 Gln Arg Leu Gln Leu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln900 905 910 gac ctg gcc tct gtg ctt agt gcc agc ccc agc ctg aag gag ctagac 2784 Asp Leu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu Lys Glu Leu Asp915 920 925 ctg cag cag aac aac ctg gat gac gtt ggc gtg cga ctg ctc tgtgag 2832 Leu Gln Gln Asn Asn Leu Asp Asp Val Gly Val Arg Leu Leu Cys Glu930 935 940 ggg ctc agg cat cct gcc tgc aaa ctc ata cgc ctg ggg aaa ccaagt 2880 Gly Leu Arg His Pro Ala Cys Lys Leu Ile Arg Leu Gly Lys Pro Ser945 950 955 960 gtg atg acc cct act gag ggc ctg gat acg gga gag atg agtaat agc 2928 Val Met Thr Pro Thr Glu Gly Leu Asp Thr Gly Glu Met Ser AsnSer 965 970 975 aca tcc tca ctc aag cgg cag aga ctc gga tca gag agg gcggct tcc 2976 Thr Ser Ser Leu Lys Arg Gln Arg Leu Gly Ser Glu Arg Ala AlaSer 980 985 990 cat gtt gct cag gct aat ctc aaa ctc ctg gac gtg agc aagatc ttc 3024 His Val Ala Gln Ala Asn Leu Lys Leu Leu Asp Val Ser Lys IlePhe 995 1000 1005 cca att gct gag att gca gag gaa agc tcc cca gag gtagta ccg gtg 3072 Pro Ile Ala Glu Ile Ala Glu Glu Ser Ser Pro Glu Val ValPro Val 1010 1015 1020 gaa ctc ttg tgc gtg cct tct cct gcc tct caa ggggac ctg cat acg 3120 Glu Leu Leu Cys Val Pro Ser Pro Ala Ser Gln Gly AspLeu His Thr 1025 1030 1035 1040 aag cct ttg ggg act gac gat gac ttt ctgggg cct gaa gga aat gtg 3168 Lys Pro Leu Gly Thr Asp Asp Asp Phe Leu GlyPro Glu Gly Asn Val 1045 1050 1055 gat gtt gag ttg att gat aag agc acaaac aga tac agc gtt tgg ttc 3216 Asp Val Glu Leu Ile Asp Lys Ser Thr AsnArg Tyr Ser Val Trp Phe 1060 1065 1070 ccc act gct ggc tgg tat ctg tggtca gcc aca ggc ctc ggc ttc ctg 3264 Pro Thr Ala Gly Trp Tyr Leu Trp SerAla Thr Gly Leu Gly Phe Leu 1075 1080 1085 gta agg gat gag gtc aca gtgacg att gcg ttt ggt tcc tgg agt cag 3312 Val Arg Asp Glu Val Thr Val ThrIle Ala Phe Gly Ser Trp Ser Gln 1090 1095 1100 cac ctg gcc ctg gac ctgcag cac cat gaa cag tgg ctg gtg ggc ggc 3360 His Leu Ala Leu Asp Leu GlnHis His Glu Gln Trp Leu Val Gly Gly 1105 1110 1115 1120 ccc ttg ttt gatgtc act gca gag cca gag gag gct gtc gcc gaa atc 3408 Pro Leu Phe Asp ValThr Ala Glu Pro Glu Glu Ala Val Ala Glu Ile 1125 1130 1135 cac ctc ccccac ttc atc tcc ctc caa ggt gag gtg gac gtc tcc tgg 3456 His Leu Pro HisPhe Ile Ser Leu Gln Gly Glu Val Asp Val Ser Trp 1140 1145 1150 ttt ctcgtt gcc cat ttt aag aat gaa ggg atg gtc ctg gag cat cca 3504 Phe Leu ValAla His Phe Lys Asn Glu Gly Met Val Leu Glu His Pro 1155 1160 1165 gcccgg gtg gag cct ttc tat gct gtc ctg gaa agc ccc agc ttc tct 3552 Ala ArgVal Glu Pro Phe Tyr Ala Val Leu Glu Ser Pro Ser Phe Ser 1170 1175 1180ctg atg ggc atc ctg ctg cgg atc gcc agt ggg act cgc ctc tcc atc 3600 LeuMet Gly Ile Leu Leu Arg Ile Ala Ser Gly Thr Arg Leu Ser Ile 1185 11901195 1200 ccc atc act tcc aac aca ttg atc tat tat cac ccc cac ccc gaagat 3648 Pro Ile Thr Ser Asn Thr Leu Ile Tyr Tyr His Pro His Pro Glu Asp1205 1210 1215 att aag ttc cac ttg tac ctt gtc ccc agc gac gcc ttg ctaaca aag 3696 Ile Lys Phe His Leu Tyr Leu Val Pro Ser Asp Ala Leu Leu ThrLys 1220 1225 1230 gcg ata gat gat gag gaa gat cgc ttc cat ggt gtg cgcctg cag act 3744 Ala Ile Asp Asp Glu Glu Asp Arg Phe His Gly Val Arg LeuGln Thr 1235 1240 1245 tcg ccc cca atg gaa ccc ctg aac ttt ggt tcc agttat att gtg tct 3792 Ser Pro Pro Met Glu Pro Leu Asn Phe Gly Ser Ser TyrIle Val Ser 1250 1255 1260 aat tct gct aac ctg aaa gta atg ccc aag gagttg aaa ttg tcc tac 3840 Asn Ser Ala Asn Leu Lys Val Met Pro Lys Glu LeuLys Leu Ser Tyr 1265 1270 1275 1280 agg agc cct gga gaa att cag cac ttctca aaa ttc tat gct ggg cag 3888 Arg Ser Pro Gly Glu Ile Gln His Phe SerLys Phe Tyr Ala Gly Gln 1285 1290 1295 atg aag gaa ccc att caa ctt gagatt act gaa aaa aga cat ggg act 3936 Met Lys Glu Pro Ile Gln Leu Glu IleThr Glu Lys Arg His Gly Thr 1300 1305 1310 ttg gtg tgg gat act gag gtgaag cca gtg gat ctc cag ctt gta gct 3984 Leu Val Trp Asp Thr Glu Val LysPro Val Asp Leu Gln Leu Val Ala 1315 1320 1325 gca tca gcc cct cct cctttc tca ggt gca gcc ttt gtg aag gag aac 4032 Ala Ser Ala Pro Pro Pro PheSer Gly Ala Ala Phe Val Lys Glu Asn 1330 1335 1340 cac cgg caa ctc caagcc agg atg ggg gac ctg aaa ggg gtg ctc gat 4080 His Arg Gln Leu Gln AlaArg Met Gly Asp Leu Lys Gly Val Leu Asp 1345 1350 1355 1360 gat ctc caggac aat gag gtt ctt act gag aat gag aag gag ctg gtg 4128 Asp Leu Gln AspAsn Glu Val Leu Thr Glu Asn Glu Lys Glu Leu Val 1365 1370 1375 gag caggaa aag aca cgg cag agc aag aat gag gcc ttg ctg agc atg 4176 Glu Gln GluLys Thr Arg Gln Ser Lys Asn Glu Ala Leu Leu Ser Met 1380 1385 1390 gtggag aag aaa ggg gac ctg gcc ctg gac gtg ctc ttc aga agc att 4224 Val GluLys Lys Gly Asp Leu Ala Leu Asp Val Leu Phe Arg Ser Ile 1395 1400 1405agt gaa agg gac cct tac ctc gtg tcc tat ctt aga cag cag aat ttg 4272 SerGlu Arg Asp Pro Tyr Leu Val Ser Tyr Leu Arg Gln Gln Asn Leu 1410 14151420 taaaatgagt cagttaggta gtctggaaga gagaatccag cgttctcatt ggaaatggat4332 aaacagaaat gtgatcattg atttcagtgt tcaagacaga agaagactgg gtaacatcta4392 tcacacaggc tttcaggaca gacttgtaac ctggcatgta cctattgact gtatcctcat4452 gcattttcct caag 4466 12 1424 PRT Artificial Sequence Description ofArtificial Sequence Synthetic 12 Met Ala Gly Gly Ala Trp Gly Arg Leu AlaCys Tyr Leu Glu Phe Leu 1 5 10 15 Lys Lys Glu Glu Leu Lys Glu Phe GlnLeu Leu Leu Ala Asn Lys Ala 20 25 30 His Ser Arg Ser Ser Ser Gly Glu ThrPro Ala Gln Pro Glu Lys Thr 35 40 45 Ser Gly Met Glu Val Ala Ser Tyr LeuVal Ala Gln Tyr Gly Glu Gln 50 55 60 Arg Ala Trp Asp Leu Ala Leu His ThrTrp Glu Gln Met Gly Leu Arg 65 70 75 80 Ser Leu Cys Ala Gln Ala Gln GluGly Ala Gly His Ser Pro Ser Phe 85 90 95 Pro Tyr Ser Pro Ser Glu Pro HisLeu Gly Ser Pro Ser Gln Pro Thr 100 105 110 Ser Thr Ala Val Leu Met ProTrp Ile His Glu Leu Pro Ala Gly Cys 115 120 125 Thr Gln Gly Ser Glu ArgArg Val Leu Arg Gln Leu Pro Asp Thr Ser 130 135 140 Gly Arg Arg Trp ArgGlu Ile Ser Ala Ser Leu Leu Tyr Gln Ala Leu 145 150 155 160 Pro Ser SerPro Asp His Glu Ser Pro Ser Gln Glu Ser Pro Asn Ala 165 170 175 Pro ThrSer Thr Ala Val Leu Gly Ser Trp Gly Ser Pro Pro Gln Pro 180 185 190 SerLeu Ala Pro Arg Glu Gln Glu Ala Pro Gly Thr Gln Trp Pro Leu 195 200 205Asp Glu Thr Ser Gly Ile Tyr Tyr Thr Glu Ile Arg Glu Arg Glu Arg 210 215220 Glu Lys Ser Glu Lys Gly Arg Pro Pro Trp Ala Ala Val Val Gly Thr 225230 235 240 Pro Pro Gln Ala His Thr Ser Leu Gln Pro His His His Pro TrpGlu 245 250 255 Pro Ser Val Arg Glu Ser Leu Cys Ser Thr Trp Pro Trp LysAsn Glu 260 265 270 Asp Phe Asn Gln Lys Phe Thr Gln Leu Leu Leu Leu GlnArg Pro His 275 280 285 Pro Arg Ser Gln Asp Pro Leu Val Lys Arg Ser TrpPro Asp Tyr Val 290 295 300 Glu Glu Asn Arg Gly His Leu Ile Glu Ile ArgAsp Leu Phe Gly Pro 305 310 315 320 Gly Leu Asp Thr Gln Glu Pro Arg IleVal Ile Leu Gln Gly Ala Ala 325 330 335 Gly Ile Gly Lys Ser Thr Leu AlaArg Gln Val Lys Glu Ala Trp Gly 340 345 350 Arg Gly Gln Leu Tyr Gly AspArg Phe Gln His Val Phe Tyr Phe Ser 355 360 365 Cys Arg Glu Leu Ala GlnSer Lys Val Val Ser Leu Ala Glu Leu Ile 370 375 380 Gly Lys Asp Gly ThrAla Thr Pro Ala Pro Ile Arg Gln Ile Leu Ser 385 390 395 400 Arg Pro GluArg Leu Leu Phe Ile Leu Asp Gly Val Asp Glu Pro Gly 405 410 415 Trp ValLeu Gln Glu Pro Ser Ser Glu Leu Cys Leu His Trp Ser Gln 420 425 430 ProGln Pro Ala Asp Ala Leu Leu Gly Ser Leu Leu Gly Lys Thr Ile 435 440 445Leu Pro Glu Ala Ser Phe Leu Ile Thr Ala Arg Thr Thr Ala Leu Gln 450 455460 Asn Leu Ile Pro Ser Leu Glu Gln Ala Arg Trp Val Glu Val Leu Gly 465470 475 480 Phe Ser Glu Ser Ser Arg Lys Glu Tyr Phe Tyr Arg Tyr Phe ThrAsp 485 490 495 Glu Arg Gln Ala Ile Arg Ala Phe Arg Leu Val Lys Ser AsnLys Glu 500 505 510 Leu Trp Ala Leu Cys Leu Val Pro Trp Val Ser Trp LeuAla Cys Thr 515 520 525 Cys Leu Met Gln Gln Met Lys Arg Lys Glu Lys LeuThr Leu Thr Ser 530 535 540 Lys Thr Thr Thr Thr Leu Cys Leu His Tyr LeuAla Gln Ala Leu Gln 545 550 555 560 Ala Gln Pro Leu Gly Pro Gln Leu ArgAsp Leu Cys Ser Leu Ala Ala 565 570 575 Glu Gly Ile Trp Gln Lys Lys ThrLeu Phe Ser Pro Asp Asp Leu Arg 580 585 590 Lys His Gly Leu Asp Gly AlaIle Ile Ser Thr Phe Leu Lys Met Gly 595 600 605 Ile Leu Gln Glu His ProIle Pro Leu Ser Tyr Ser Phe Ile His Leu 610 615 620 Cys Phe Gln Glu PhePhe Ala Ala Met Ser Tyr Val Leu Glu Asp Glu 625 630 635 640 Lys Gly ArgGly Lys His Ser Asn Cys Ile Ile Asp Leu Glu Lys Thr 645 650 655 Leu GluAla Tyr Gly Ile His Gly Leu Phe Gly Ala Ser Thr Thr Arg 660 665 670 PheLeu Leu Gly Leu Leu Ser Asp Glu Gly Glu Arg Glu Met Glu Asn 675 680 685Ile Phe His Cys Arg Leu Ser Gln Gly Arg Asn Leu Met Gln Trp Val 690 695700 Pro Ser Leu Gln Leu Leu Leu Gln Pro His Ser Leu Glu Ser Leu His 705710 715 720 Cys Leu Tyr Glu Thr Arg Asn Lys Thr Phe Leu Thr Gln Val MetAla 725 730 735 His Phe Glu Glu Met Gly Met Cys Val Glu Thr Asp Met GluLeu Leu 740 745 750 Val Cys Thr Phe Cys Ile Lys Phe Ser Arg His Val LysLys Leu Gln 755 760 765 Leu Ile Glu Gly Arg Gln His Arg Ser Thr Trp SerPro Thr Met Val 770 775 780 Val Leu Phe Arg Trp Val Pro Val Thr Asp AlaTyr Trp Gln Ile Leu 785 790 795 800 Phe Ser Val Leu Lys Val Thr Arg AsnLeu Lys Glu Leu Asp Leu Ser 805 810 815 Gly Asn Ser Leu Ser His Ser AlaVal Lys Ser Leu Cys Lys Thr Leu 820 825 830 Arg Arg Pro Arg Cys Leu LeuGlu Thr Leu Arg Leu Ala Gly Cys Gly 835 840 845 Leu Thr Ala Glu Asp CysLys Asp Leu Ala Phe Gly Leu Arg Ala Asn 850 855 860 Gln Thr Leu Thr GluLeu Asp Leu Ser Phe Asn Val Leu Thr Asp Ala 865 870 875 880 Gly Ala LysHis Leu Cys Gln Arg Leu Arg Gln Pro Ser Cys Lys Leu 885 890 895 Gln ArgLeu Gln Leu Val Ser Cys Gly Leu Thr Ser Asp Cys Cys Gln 900 905 910 AspLeu Ala Ser Val Leu Ser Ala Ser Pro Ser Leu Lys Glu Leu Asp 915 920 925Leu Gln Gln Asn Asn Leu Asp Asp Val Gly Val Arg Leu Leu Cys Glu 930 935940 Gly Leu Arg His Pro Ala Cys Lys Leu Ile Arg Leu Gly Lys Pro Ser 945950 955 960 Val Met Thr Pro Thr Glu Gly Leu Asp Thr Gly Glu Met Ser AsnSer 965 970 975 Thr Ser Ser Leu Lys Arg Gln Arg Leu Gly Ser Glu Arg AlaAla Ser 980 985 990 His Val Ala Gln Ala Asn Leu Lys Leu Leu Asp Val SerLys Ile Phe 995 1000 1005 Pro Ile Ala Glu Ile Ala Glu Glu Ser Ser ProGlu Val Val Pro Val 1010 1015 1020 Glu Leu Leu Cys Val Pro Ser Pro AlaSer Gln Gly Asp Leu His Thr 1025 1030 1035 1040 Lys Pro Leu Gly Thr AspAsp Asp Phe Leu Gly Pro Glu Gly Asn Val 1045 1050 1055 Asp Val Glu LeuIle Asp Lys Ser Thr Asn Arg Tyr Ser Val Trp Phe 1060 1065 1070 Pro ThrAla Gly Trp Tyr Leu Trp Ser Ala Thr Gly Leu Gly Phe Leu 1075 1080 1085Val Arg Asp Glu Val Thr Val Thr Ile Ala Phe Gly Ser Trp Ser Gln 10901095 1100 His Leu Ala Leu Asp Leu Gln His His Glu Gln Trp Leu Val GlyGly 1105 1110 1115 1120 Pro Leu Phe Asp Val Thr Ala Glu Pro Glu Glu AlaVal Ala Glu Ile 1125 1130 1135 His Leu Pro His Phe Ile Ser Leu Gln GlyGlu Val Asp Val Ser Trp 1140 1145 1150 Phe Leu Val Ala His Phe Lys AsnGlu Gly Met Val Leu Glu His Pro 1155 1160 1165 Ala Arg Val Glu Pro PheTyr Ala Val Leu Glu Ser Pro Ser Phe Ser 1170 1175 1180 Leu Met Gly IleLeu Leu Arg Ile Ala Ser Gly Thr Arg Leu Ser Ile 1185 1190 1195 1200 ProIle Thr Ser Asn Thr Leu Ile Tyr Tyr His Pro His Pro Glu Asp 1205 12101215 Ile Lys Phe His Leu Tyr Leu Val Pro Ser Asp Ala Leu Leu Thr Lys1220 1225 1230 Ala Ile Asp Asp Glu Glu Asp Arg Phe His Gly Val Arg LeuGln Thr 1235 1240 1245 Ser Pro Pro Met Glu Pro Leu Asn Phe Gly Ser SerTyr Ile Val Ser 1250 1255 1260 Asn Ser Ala Asn Leu Lys Val Met Pro LysGlu Leu Lys Leu Ser Tyr 1265 1270 1275 1280 Arg Ser Pro Gly Glu Ile GlnHis Phe Ser Lys Phe Tyr Ala Gly Gln 1285 1290 1295 Met Lys Glu Pro IleGln Leu Glu Ile Thr Glu Lys Arg His Gly Thr 1300 1305 1310 Leu Val TrpAsp Thr Glu Val Lys Pro Val Asp Leu Gln Leu Val Ala 1315 1320 1325 AlaSer Ala Pro Pro Pro Phe Ser Gly Ala Ala Phe Val Lys Glu Asn 1330 13351340 His Arg Gln Leu Gln Ala Arg Met Gly Asp Leu Lys Gly Val Leu Asp1345 1350 1355 1360 Asp Leu Gln Asp Asn Glu Val Leu Thr Glu Asn Glu LysGlu Leu Val 1365 1370 1375 Glu Gln Glu Lys Thr Arg Gln Ser Lys Asn GluAla Leu Leu Ser Met 1380 1385 1390 Val Glu Lys Lys Gly Asp Leu Ala LeuAsp Val Leu Phe Arg Ser Ile 1395 1400 1405 Ser Glu Arg Asp Pro Tyr LeuVal Ser Tyr Leu Arg Gln Gln Asn Leu 1410 1415 1420 13 32 DNA ArtificialSequence Description of Artificial Sequence Primer 13 ccgaattcaccatggctggc ggagcctggg gc 32 14 34 DNA Artificial Sequence Description ofArtificial Sequence Primer 14 ccgctcgagt caacagaggg ttgtggtggt cttg 3415 31 DNA Artificial Sequence Description of Artificial Sequence Primer15 cccgaattcg aacctcgcat agtcatactg c 31 16 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 gtcccacaac agaattcaatctcaacggtc 30 17 21 DNA Homo sapiens 17 tgtgatgaga gaagcggtga c 21 18 30DNA Artificial Sequence Description of Artificial Sequence Primer 18ccgctcgagc aaagaagggt cagccaaagc 30

That which is claimed is:
 1. Isolated nucleic acid encoding a NB-ARC andCARD containing protein (NAC), or functional fragments thereof, selectedfrom: (a) DNA encoding the amino acid sequence set forth in SEQ IDNOs:2, 4 or 6, or (b) DNA that hybridizes to the DNA of (a) undermoderately stringent conditions, wherein said DNA encodes biologicallyactive NAC, or (c) DNA degenerate with respect to either (a) or (b)above, wherein said DNA encodes biologically active NAC.
 2. A nucleicacid according to claim 1, wherein said nucleic acid hybridizes underhigh stringency conditions to the NAC coding portion of any of SEQ IDNOs:1, 3 and
 5. 3. A nucleic acid according to claim 1, wherein thenucleotide sequence of said nucleic acid is substantially the same asset forth in any of SEQ ID NO:1, 3 and
 5. 4. A nucleic acid according toclaim 1, wherein the nucleotide sequence of said nucleic acid is thesame as that set forth in any of SEQ ID NOs:1, 3 and
 5. 5. A nucleicacid according to claim 1, wherein said nucleic acid is cDNA.
 6. Avector containing the nucleic acid of claim
 1. 7. Recombinant cellscontaining the nucleic acid of claim
 1. 8. An oligonucleotide comprisingat least 15 nucleotides capable of specifically hybridizing with a thenucleotide sequence set forth in any of SEQ ID NOs:1, 3 and
 5. 9. Anoligonucleotide according to claim 8, wherein said oligonucleotide islabeled with a detectable marker.
 10. An antisense-nucleic acid capableof specifically binding to mRNA encoded by said nucleic acid accordingto claim
 1. 11. A kit for detecting the presence of the NAC cDNAsequence comprising at least one oligonucleotide according to claim 9.12. An isolated NAC protein comprising a NB-ARC domain, a CARD domainand a TIM-Barrel-like domain.
 13. The protein of claim 12, furthercomprising a LRR domain.
 14. An isolated protein according to claim 12,wherein the amino acid sequence of said protein comprises substantiallythe same sequence as any of SEQ ID NOs:2, 4 or
 6. 15. A NAC according toclaim 14 comprising the same amino acid sequence as set forth in any ofSEQ ID NOs:2, 4 or
 6. 16. A NAC according to claim 14, wherein saidprotein is encoded by a nucleotide sequence comprising substantially thesame nucleotide sequence as set forth in SEQ ID NOs:1, 3 or
 5. 17. A NACaccording to claim 14, wherein said protein is encoded by a nucleotidesequence comprising the same sequence as set forth in SEQ ID NOs:1, 3 or5.
 18. A method for expression of a NAC protein, said method comprisingculturing cells of claim 7 under conditions suitable for expression ofsaid NAC.
 19. An isolated anti-NAC antibody having specific reactivitywith a NAC according to claim
 12. 20. Antibody according to claim 19,wherein said antibody is a monoclonal antibody.
 21. A cell lineproducing the monoclonal antibody of claim
 20. 22. An antibody accordingto claim 19, wherein said antibody is a polyclonal antibody.
 23. Acomposition comprising an amount of the antisense-nucleic acid accordingto claim 10 effective to inhibit expression of a human NAC and anacceptable hydrophobic carrier capable of passing through a cellmembrane.
 24. A transgenic nonhuman mammal expressing exogenous nucleicacid according to claim 1, encoding a NAC.
 25. A transgenic nonhumanmammal according to claim 24, wherein said nucleic acid encoding saidNAC has been mutated, and wherein the NAC so expressed is not nativeNAC.
 26. A transgenic nonhuman mammal according to claim 24, wherein thetransgenic nonhuman mammal is a mouse.
 27. A method for identifyingnucleic acids encoding a mammalian NAC, said method comprising:contacting a sample containing nucleic acids with an oligonucleotideaccording to claim 8, wherein said contacting is effected under highstringency hybridization conditions, and identifying compounds whichhybridize thereto.
 28. A method for detecting the presence of a humanNAC in a sample, said method comprising contacting a test sample with anantibody according to claim 19, detecting the presence of anantibody:NAC complex, and therefor detecting the presence of a human NACin said test sample.
 29. Single strand DNA primers for amplification ofNAC nucleic acid, wherein said primers comprise a nucleic acid sequencederived from the nucleic acid sequences set forth as SEQ ID NOs:1, 3 and5.
 30. A method for modulating the activity of an oncogenic protein,comprising contacting said oncogenic proteins with a substantially pureNAC, or an oncogenic protein-binding fragment thereof.
 31. A method ofidentifying an effective agent that alters the association of a NAC witha NAC associated protein (NAP), comprising the steps of: a) contactingsaid NAC and NAP proteins, under conditions that allow said NAC and NAPproteins to associate with an agent suspected of being able to alter theassociation of said NAC and NAP proteins; and b) detecting the alteredassociation of said NAC and NAP proteins, wherein said alteredassociation identifies an effective agent.
 32. The method of claim 31,wherein said altered association is detected by measuring thetranscriptional activity of a reporter gene.
 33. The method of claim 31,wherein said NAC has nucleotide binding activity.
 34. The method ofclaim 31, wherein said effective agent is a drug.
 35. The method ofclaim 31, wherein said effective agent is a protein.
 36. A method formodulating an activity mediated by a NAC protein, said methodcomprising: contacting said NAC protein with an effective, modulatingamount of an agent identified by claim
 31. 37. The method of claim 36,wherein said modulated activity is selected from the group consistingof: binding of NAC to a CARD-containing protein; binding of NAC to aNB-ARC-containing protein; binding of NAC to a LRR-containing protein;and caspase proteolytic activity.
 38. A method of modulating the levelapoptosis in a cell, comprising the steps of: a) introducing a nucleicacid molecule encoding a NAC into the cell; and b) expressing said NACin said cell, wherein the expression of said NAC modulates apoptosis insaid cell.
 39. A method of modulating the level of apoptosis in a cell,comprising introducing an antisense nucleotide sequence into the cell,wherein said antisense nucleotide sequence specifically hybridizes to anucleic acid molecule encoding a NAC, wherein said hybridization reducesor inhibits the expression of said NAC in said cell.
 40. A therapeuticcomposition comprising a compound selected from a NAC, or functionalfragment thereof, a NAC modulating agent identified according to claim31, or an anti-NAC antibody; and a pharmaceutically acceptable carrier.41. A method of treating a pathology characterized by abnormal cellproliferation or abnormal inflammation, said method comprisingadministering an effective amount of the composition according to claim40.
 42. A method of diagnosing a pathology characterized by an increasedor decreased level of a NAC in a subject, comprising the steps of: a)obtaining a test sample from the subject; b) contacting said test samplewith an agent that can bind said NAC under suitable conditions, whichallow specific binding of said agent to said NAC; and c) comparing theamount of said specific binding in said test sample with the amount ofspecific binding in a control sample, wherein an increased or decreasedamount of said specific binding in said test sample as compared to saidcontrol sample is diagnostic of a pathology.
 43. The method of claim 42,wherein said agent is an anti-NAC antibody or a NAC-associated-protein(NAP).
 44. A method of modulating the level of apoptosis in a cell,comprising contacting the cell with an agent that effectively alters theassociation of NAC with a NAC-associated-protein in the cell, or thateffectively alters the activity of a NAC in the cell.
 45. A chimericprotein comprising a domain selected from the group consisting of theNB-ARC domain of the NAC of claim 14 and the CARD of the NAC of claim14.
 46. An isolated protein comprising a TIM-Barrel-like domain and asecond domain selected from the group consisting of a CARD domain, aNB-ARC domain, and a LRR domain.
 47. The chimeric protein of claim 45,comprising the NB-ARC domain of SEQ ID NO:2 and the CARD domain of SEQID NO:8.
 48. The method of claim 31, wherein said agent modulatesCARD:CARD association or NB-ARC:NB-ARC association.
 49. A method ofmodulating CARD:CARD interactions comprising contacting a NAC proteinwith the agent of claim
 48. 50. The method of claim 31, wherein saidagent modulates transcription.
 51. The method of claim 50, wherein saidagent modulates NF-κB activity.
 52. A method of modulating transcriptioncomprising contacting a cell with a compound selected from the groupconsisting of: a NAC protein or functional fragment thereof, an agentidentified according to claim 31, and an anti-NAC antibody.
 53. A methodof diagnosing cancer or monitoring cancer therapy comprising contactinga test sample from a patient with the antibody of claim
 19. 54. A methodof assessing prognosis of patients with cancer comprising contacting atest sample from a patient with the antibody of claim
 19. 55. Aneffective agent that binds a nucleotide binding site of NAC.
 56. Aneffective agent that modulates the association of NAC or CARD-X with apro-caspase or a caspase.
 57. The method of claim 56, wherein saidpro-caspase is pro-caspase-8 and said caspase is caspase-8.
 58. Themethod of claim 56, wherein said pro-caspase is pro-caspase-9 and saidcaspase is caspase-9.
 59. The method of claim 56, wherein said effectiveagent inhibits the association of said NAC with said pro-caspase or saidcaspase.
 60. The method of claim 56, wherein said effective agentincreases the association of said NAC with said pro-caspase or saidcaspase.
 61. An effective agent that modulates the association of NAC orCARD-X with a CED-4 family protein.
 62. The method of claim 61, whereinsaid CED-4 family protein is selected from the group consisting ofCED-4, Apaf-1, Dark, and CARD4/nod1.
 63. The method of claim 61, whereinsaid CED-4 family protein is Apaf-1.
 64. The method of claim 61, whereinsaid effective agent inhibits the association of said NAC with saidCED-4 family protein.
 65. The method of claim 61, wherein said effectiveagent increases the association of said NAC with said CED-4 familyprotein.